Staphylokinase derivatives with cysteine substitutions

ABSTRACT

Methods for the identification, production and use of staphylokinase derivatives characterized by a reduced immunogenicity after administration in patients. The derivatives of the invention are obtained by preparing a DNA fragment comprising at least the part of the coding sequence of staphylokinase that provides for its biological activity; performing in vitro site-directed mutagenesis on the DNA fragment to replace one or more codons for wild-type amino acids by a codon for another amino acid; cloning the mutated DNA fragment in a suitable vector; transforming or transfecting a suitable host cell with the vector; culturing the host cell under conditions suitable for expressing the DNA fragment; and purifying the expressed staphylokinase derivative to homogeneity. Preferably the DNA fragment is a 453 bp EcoRI-HindIII fragment of the plasmid pMEX602sakB, (pMEX.SakSTAR), the in vitro site-directed mutagenesis is performed by spliced overlap extension polymerase chain reaction and the mutated DNA fragment is expressed in  E. coli  strain TG1 or WK6. The invention also relates to pharmaceutical compositions comprising at least one of the staphylokinase derivatives according to the invention together with a suitable excipient, for treatment of arterial thrombosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/784,971, filed Jan. 16, 1997, now U.S. Pat. No. 5,951,980which is a continuation-in-part of U.S. patent application Ser. No.08/499,092, filed Jul. 6, 1995, now abandoned which is acontinuation-in-part of U.S. patent application Ser. No. 08/371,505,filed Jan. 11, 1995, now U.S. Pat. No. 5,695,754, issued Dec. 9, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to new staphylokinase derivatives with reducedimmunogenicity, their identification, production and use in thetreatment of arterial thrombosis and the preparation of a pharmaceuticalcomposition for treating arterial thrombosis. More in particular itrelates to the use of engineered staphylokinase derivatives for thepreparation of a pharmaceutical composition for treating myocardialinfarction.

2. Description of the Related Art

Staphylokinase, a protein produced by certain strains of Staphylococcusaureus, which was shown to have profibrinolytic properties more than 4decades ago (1,2) appears to constitute a potent thrombolytic agent inpatients with acute myocardial infarction (3,4). The staphylokinase genehas been cloned from the bacteriophages sakøC (5) and sak42D (6) as wellas from the genomic DNA (sakSTAR) of a lysogenic Staphylococcus aureusstrain (7). The staphylokinase gene encodes a protein of 163 aminoacids, with amino acid 28 corresponding to the NH₂-terminal residue offull length mature staphylokinase (6,8,9). The mature protein sequenceof the wild-type variant SakSTAR (9) is represented in FIG. 1. Only fournucleotide differences were found in the coding regions of the sakøC,sak42D and sakSTAR genes, one of which constituted a silent mutation(6,8,9).

SUMMARY OF THE INVENTION

In a plasma milieu, staphylokinase is able to dissolve fibrin clotswithout associated fibrinogen degradation (10-12). Thisfibrin-specificity of staphylokinase is the result of reduced inhibitionby α₂-antiplasmin of plasmin.staphylokinase complex bound to fibrin,recycling of staphylokinase from the plasmin.staphylokinase complexfollowing inhibition by α₂-antiplasin, and prevention of the conversionof circulating plasminogen.staphylokinase to plasmin.staphylokinase byα₂-antiplasmin (13-15). In addition staphylokinase has a weak affinityfor circulating but a high affinity for fibrin-bound plasminogen (16)and staphylokinase requires NH₂-terminal processing by plasmin todisplay its plasminogen activating potential (17). In severalexperimental animal models, staphylokinase appears to be equipotent tostreptokinase for the dissolution of whole blood or plasma clots, butsignificantly more potent for the dissolution of platelet-rich orretracted thrombi (18,19).

Staphylokinase is a heterologous protein and is immunogenic in man. Theintrinsic immunogenicity of staphylokinase, like that of streptokinase,clearly hampers its unrestricted use. Not only will patients withpreexisting high antibody titers be refractory to the thrombolyticeffect of these agents, but allergic side effects and occasionallife-threatening anaphylaxis may occur (20). Because both streptokinaseand staphylokinase are heterologous proteins, it is not obvious thattheir inmmunogenicity could be reduced by protein engineering. Indeed,no successful attempts to generate active low molecular weight fragmentsfrom streptokinase have been reported. In staphylokinase, deletion ofthe NH₂-terminal 17 amino acids or the COOH-terminal 2 amino acidsinactivates the molecule, which in addition is very sensitive toinactivation by site-specific mutagenesis (21).

Nevertheless, we have, surprisingly, found that the wild-typestaphylokinase variant SakSTAR (9) contains three non-overlappingimmunodominant epitopes, at least two of which can be eliminated byspecific site-directed mutagenesis, without inactivation of the molecule(22). These engineered staphylokinase variants are less reactive withantibodies elicited in patients treated with wild-type staphylokinase,and are significantly less immunogenic than wild-type staphylokinase, asdemonstrated in rabbit and baboon models and in patients with peripheralarterial occlusion (22).

The present invention relates to general methods for the identification,production and use of staphylokinase derivatives showing a reducedantigenicity and immunogenicity as compared to wild-type staphylokinase.The derivatives have essentially the amino acid sequence of wild-typestaphylokinase or modified versions thereof and essentially intactbiological activities, but have a reduced reactivity with a panel ofmurine monoclonal antibodies and/or with antibodies induced in patientsby treatment with wild-type SakSTAR.

The invention also relates to a method for producing the derivatives ofthe invention by preparing a DNA fragment comprising at least the partof the coding sequence of staphylokinase that provides for itsbiological activity; performing in vitro site-directed mutagenesis onthe DNA fragment to replace one or more codons for wild-type amino acidsby a codon for another amino acid; cloning the mutated DNA fragment in asuitable vector; transforming or transfecting a suitable host cell withthe vector; culturing the host cell under conditions suitable forexpressing the DNA fragment and purifying the expressed staphylokinasederivative to homogeneity; preferably the DNA fragment is a 453 bpEcoRI-HindIII fragment of the plasmid pMEX602sakB (22,23), the in vitrosite-directed mutagenesis is preferably performed by spliced overlapextension polymerase chain reaction with Vent DNA polymerase (NewEngland Biolabs) or Taq polymerase (Boehringer Mannheim) and withavailable or generated wildtype sakSTAR or sakSTAR variants as template(24).

The invention also relates to pharmaceutical compositions comprising atleast one of the staphylokinase derivatives according to the inventiontogether with a suitable excipient, for treatment of arterialthrombosis. Pharmaceutical compositions, containing less immunogenicstaphylokinase variants as the active ingredient, for treating arterialthrombosis in human or veterinary practice may take the form of powdersor solutions and may be used for intravenous, intraarterial orparenteral administration. Such compositions may be prepared bycombining (e.g. mixing, dissolving etc.) the active compound withpharmaceutically acceptable excipients of neutral character (such asaqueous or non-aqueous solvents, stabilizers, emulsifiers, detergents,additives), and further, if necessary with dyes.

Furthermore the invention relates to the use of the staphylokinasederivatives for the treatment of arterial thrombosis, in particularmyocardial infarction, and to the use of staphylokinase derivatives forthe preparation of a pharmaceutical composition for the treatment ofarterial thrombosis, in particular myocardial infarction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a protein sequence of wild-type staphylokinase, SakSTAR (SEQID NO: 10). Numbering starts with the NH₂-terminal amino acid of maturefull length staphylokinase.

FIG. 2 is a time course of neutralizing activities (left panel) andspecific IgG against administered agent (right panel) followingintra-arterial infusion of SakSTAR (open circles, n=9), SakSTAR(K74A)(closed circles, n=11) or SakSTAR(K74A, E75A, R77A) (open squares, n=6)in patients with peripheral arterial occlusion. The data representmedian values and interquartile ranges, in μg/ml.

FIG. 3 is a protein sequence of wild-type staphylokinase, SakSTAR withindicated amino acid substitutions.

Squares: single amino acid substitutions; circles: combined (2 to 3)amino acid to Ala substitutions.

FIG. 4 shows temperature stability of SakSTAR, (A); SakSTAR (K74Q, E80A,D82A, K130T, K135R), (B); SakSTAR (E65D, K74R, E80A, D82A, K130T,K135R), (C); and SakSTAR (K35A, E65D, K74Q, E80A, D82A, K130T, K135R),(D).

(◯): 4° C.; (): 20° C.; (∇): 37° C.; (▾): 56° C.; (□): 70° C.

FIG. 5 is a time course of neutralizing activities left panel) andspecific IgG against administered agent (right panel) followingintra-arterial infusion of SakSTAR (circles, n=15), SakSTAR (K74Q, E80A,D82A, K130T, K135R) (squares, n=6) or SakSTAR (E65D, K74R, E80A, D82A,K130T, K135R) (triangles, n=6) in patients with peripheral arterialocclusion. The data represent median values and 15-85 percentile ranges,in μg/mL.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the above and the following the terms “derivatives”, “mutants” and“variants” are used interchangeably.

The present invention will be demonstrated in more detail in thefollowing examples, that are however not intended to be limiting to thescope of the invention. Based on the present invention other variantsand improvements will be obvious for the person skilled in the art. Thusrandom mutagenesis is likely to generate alternative mutants withreduced immunogenicity and possibly increased functional activity,whereas deletions or substitution with other amino acids may yieldadditional variants with reduced immunogenicity.

EXAMPLE 1

Epitope Mapping of Wild-type Staphylokinase

The epitope specificity of a panel of 15 murine MAbs (22) raised againstwild-type SakSTAR was determ ined by real-time biospecific int eractionanalysis (BIA) with the BIAcore instrument (Pharmacia, Biosensor AB,Uppsala, Sweden). The MAbs were immobilized on the surface of the SensorChip CM5 with the Amine Coupling Kit (Pharmacia Biosensor AB) asrecommended by the manufacturer (25). Immobilization was performed fromprotein solutions at a concentration of 20 μg/mL in 10 mmol/L sodiumacetate at pH 5.0 at a flow rate of 5 μL/min during 6 minutes. Thisresulted in covalent attachment of 5,000 to 10,000 resonance unit (RU)of antibody (corresponding to 0.035 to 0.07 pmol/mm²) . The SakSTARsolutions were passed by continuous flow at 20° C. past the sensorsurface. At least four concentrations of each analyte (range, 50 nmol/Lto 50 μmol/L) in 10 mmol/L HEPES, 3.4 mmol/L EDTA, 0.15 mol/L NaCl, and0.005% Surfactant P20, pH 7.2, were injected at a flow rate of 5 μL/minduring 6 minutes in the association phase. Then sample was replaced bybuffer, also at a flow rate of 5 μL/min during 6 minutes. After eachcycle, the surface of the sensor chip was regenerated by injection of 5μL of 15 mmol/L HCl. Apparent association (k_(ass)) and apparentdissociation (k_(diss)) rate constants were derived from the sensorgramsas described in detail elsewhere (26), and association equilibriumconstants (K_(A)) calculated as their ratio.

Determination of the equilibrium association constants for the bindingof wild-type and variant SakSTAR to insolubilized MAbs (Table 1) yieldedapparent association constants of 10⁷ to 10⁸ (mol/L)⁻¹, which are one totwo orders of magnitude lower than the apparent association constantspreviously obtained for the binding of these MAbs to insolubilizedwild-type SakSTAR (22). If the MAbs instead of the SakSTAR variants areinsolubilized, avidity effects of the bivalent MAbs are indeed avoided.The present values are indeed in better agreement with known associationconstants of Mabs, and therefore this “reversed” procedure was usedthroughout the present invention.

In the tables the column indicated with “Variant” states the variousstaphylokinase derivatives which are identified by listing betweenbrackets the substituted amino acids in single letter symbols followedby their position number in the mature staphylokinase sequence and bythe substituting amino acids in single letter symbol; the column “Exp.”indicates expression levels in mg/L, and the column “Spec. Act.”indicates the specific activity in Home Units as defined in example 2.Indications “17G11”, “26A2” etc. refer to monoclonal antibodies bindingto the indicated epitopes I, II and III (22). Epitope I is recognized bythe antibody cluster 17G11, 26A2, 30A2, 2B12 and 3G10, whereas epitopeII is recognized by the antibody cluster 18F12, 14H5, 28H4, 32B2 and7F10, and epitope III by the antibody cluster 7H11, 25E1, 40C8, 24C4 and1A10. Human plasma “Pool” indicates a plasma pool from initially 16 andsubsequently 10 patients immunized by treatment with SakSTAR, “SubpoolB” indicates a plasma pool from three patients that absorbed less than50% of the induced antibodies with SakSTAR(K35A,E38A,K74A,E75A,R77A) and“Subpool C” indicates a plasma pool from 3 patients that absorbed >90%of the induced antibodies with SakSTAR(K35A,E38A,K74A,E75A,R77A) (22).In tables 6, 7 and 8 an additional pool of plasma from 40 patientsimmunized by treatment with SakSTAR (Pool 40) was also used.

EXAMPLE 2

Construction, Epitope Mapping with Murine Monoclonal Antibodies andAbsorption with Pooled Plasma of Immunized Patients, of“Alanine-to-wild-type” Reversal Variants of “Charged-cluster-to-alanine”Mutants of Staphylokinase

As stated above, wild-type staphylokinase (SakSTAR variant (9)) containsthree non-overlapping immunodorninant epitopes, two of which can beeliminated by specific site-directed substitution of clusters of two(K35A,E38A or E80A,D82A) or three (K74A,E75A,R77A) charged amino acidswith Ala (22). The combination mutants SakSTAR(K35A,E38A,K74A,E75A,R77A)in which Lys35, Glu38, Lys74, Glu75 and Arg77, andSakSTAR(K74A,E75A,R77A,E80A,D82A) in which Lys74, Glu75, Arg77, Glu80and Asp82 were substituted with Ala (previously identified asSakSTAR.M3.8 and SakSTAR.M8.9, respectively (22)), were found to have areduced reactivity with murine monoclonal antibodies against two of thethree immunodominant epitopes and to absorb on average only 2/3 of theneutralizing antibodies elicited in 16 patients by treatment withwild-type SakSTAR (22). These mutants also induced less antibodyformation than wild-type SakSTAR in experimental thrombolysis models inrabbits and baboons, and in patients with peripheral arterial occlusion(22). However, their specific activities were reduced to approximately50% of that of wild-type SakSTAR, which would be of some concern withrespect to the clinical use of these compounds.

In an effort to improve the activity and stability without loss of thereduced antibody recognition, the effect of a systematic reversal of oneor more of these substituted amino acids to the wild-type residues wasstudied. Fourteen new mutants were constructed, purified andcharacterized in terms of specific activity, reactivity with the panelof murine monoclonal antibodies, and absorption of antibodies fromplasma of patients treated with wild-type SakSTAR (Table 1). The presentexample thus focusses on reversal from alanine to the wild-type residueof one or more of the seven amino acids of SakSTAR listed above i.e.K35, E38, K74, E75, R77, E80 and D82.

Reagents and Methods

The source of all reagents used in the present study has previously beenreported (22). Restriction enzymes were purchased from Pharmacia(Uppsala, Sweden) or Boehringer Mannheim (Mannheim, Germany). T4 DNAligase, Klenow Fragment of E. coli DNA polymerase I and alkalinephosphatase were obtained from Boehringer Mannheim. Enzyme reactionswere performed using the conditions suggested by the suppliers. PlasmidDNA was isolated using a QIAGEN-purification protocol (provided byWestburg, Leusden, The Netherlands). pMEX.602sakB (i.e. pMEX.SakSTAR)was constructed as described elsewhere (23). SakSTAR,SakSTAR(K35A,E38A), SakSTAR(K74A,E75A,R77A), SakSTAR(E80A,D82A),SakSTAR(K35A,E38A,K74A,E75A,R77A) and SakSTAR(K74A,E75A,R77A,E80A,D82A)were produced and purified as described elsewhere (22). Transformationsof E. coli were performed utilizing the calcium phosphate procedure. DNAsequencing was performed using the dideoxy chain termination reactionmethod and the Automated Laser fluorescent A.L.F.™ (Pharmacia). Thechromogenic substrate (S2403)L-Pyroglutamyl-L-phenylalanyl-L-lysine-p-nitroanaline hydrochloride waspurchased from Chromogenix (Belgium). ¹²⁵I-labeled fibrinogen waspurchased from Amersham (UK). All other methods used in the presentexample have been previously described (22,27).

Construction of Expression Plasmids

The plasmids encoding SakSTAR(K35A,E38A,K74A,E75A),SakSTAR(E38A,E75A,R77A), SakSTAR(E38A,E75A), SakSTAR(K35A,E75A,R77A),SakSTAR(K35A,E75A), SakSTAR(E80A), SakSTAR(D82A), SakSTAR(E75A,D82A),SakSTAR(K74A) and SakSTAR(E75A) were constructed by the spliced overlapextension polymerase chain reaction (SOE-PCR) (24), using Vent DNApolymerase (New England Biolabs, Leusden, The Netherlands), andavailable or generated sakSTAR variants as template. Two fragments wereamplified by PCR, the first one starting from the 5′ end of thestaphylokinase gene with primer 5′-CAGGAAACAGAATTCAGGAG-3′ (SEQ ID NO:1)to the region to be mutagenized (forward primer), the second one fromthe same region (backward primer) to the 3′ end of the sraphylokinasegene with primer 5′-CAAAACAGCCAAGCTTCATTCATTCAGC-3′ (SEQ ID NO:2). Theforward and backward primers shared an overlap of around 24 bp (primersnot shown) The two purified fragments were then assembled together in anew primeness PCR using Taq polymerase (Boehringer Mannheim). After 7cycles (1 min at 94° C., 1 min at 70° C.), the extended product wasreamplified by adding the 5′ and 3′ end primers (see above) to the PCRreaction and by cycling 25 times (1 min at 94° C, 1 min 55° C, 1 min at72° C.). The final product was purified, digested with EcoRI and HindIIIand cloned into the corresponding sites of pMEX602sakB.

The plasmid encoding SakSTAR(E38A,K74A,E75A,R77A) was assembled bydigestion of pMEX602sakB and pMEX.SakSTAR(K35A,E38A,K74A,E75A,R77A) withBpm I which cuts between the codons for K35 and E38 of SakSTAR, andligation of the required fragments. The plasmid encodingSakSTAR(K35A,K74A,E75A,R77A) was constructed by digestion ofpMEX.SakSTAR(K35A,E38A,K74A,E75A,R77A) and pMEX. SakSTAR(K74A,E75A,R77A) with Bpm I and religation of the required fragments. Theplasmids encoding SakSTAR(K35A,E38A,E75A,R77A) andSakSTAR(K35A,E38A,K74A,R77A) were constructed by two PCR usingpMEX.SakSTAR(K35A,E38A,K74A,E75A,R77A) as template, followed byrestriction ligation and recloning into pMEX602sakB.

Expression and Purification of SakSTAR Variants

The SakSTAR variants were expressed and purified, as described below,from transformed E. coli WK6 grown either in LB medium[SakSTAR(E38A,K74A,E75A,R77A), SakSTAR(K74A), SakSTAR(E75A) andSakSTAR(E75A,D82A)], or in terrific broth (TB) (28) medium[SakSTAR(K35A,K74A,E75A,R77A), SakSTAR(K35A,E38A,E75A,R77A),SakSTAR(K35A,E38A,K74A,R77A), SakSTAR(K35A,E38A,E75A), SakSTAR(E38A,E75A,R77A), SakSTAR(E38A,E75A), SakSTAR(K35A,E75A,R77A), SakSTAR(K35A,E75A), SakSTAR(E80A), and SakSTAR(D82A)].

For derivatives produced in LB medium, a 20 mL aliquot of an overnightsaturated culture was used to inoculate a 2 L volume of LB mediumcontaining 100 μg/mL ampicillin. After 3 hours incubation at 37° C.,IPTG (200 μmol/L) was added to induce expression from the tac promoter.The production phase was allowed to proceed for 4 hours, after which thecells were pelleted by centrifugation at 4,000 rpm for 20 min,resuspended in 1/20 volume (100 mL) of 0.01 mol/L phosphate buffer pH6.5 and disrupted by sonication at 0° C. Cell debris were removed bycentrifugation for 20 min at 20,000 rpm and the supernatant, containingthe cytosolic soluble protein fraction, was stored at −20° C. untilpurification.

For the derivatives produced in TB medium, a 4 mL aliquot of anovernight saturated culture in LB medium was used to inoculate a 2 Lculture in terrific broth containing 100 μg/mL ampicillin. The culturewas grown with vigorous aeration for 20 hours at 30° C. The cells werepelleted by centrifugation, resuspended in 1/10 volume (200 mL) of 0.01mol/L phosphate buffer pH 6.5 and disrupted by sonication at 0° C. Thesuspension was then centrifuged for 20 min at 20,000 rpm and thesupernatant was stored at −20° C. until purification.

Cleared cell lysates containing the SakSTAR variants were subjected tochromatography on a 1.6×6 cm column of SP-Sephadex, followed bychromatography on a 1.6×5 cm column of Q-Sepharose [variantsSakSTAR(E38A,K74A,E75A,R77A), SakSTAR(K35A,K74A,E75A, R77A),SakSTAR(K35A,E38A,E75A,R77A), SakSTAR(K35A,E38A, K74A,R77A) andSakSTAR(K35A,E38A,K74A,E75A)] or by chromatography on a 1.6×6 cm columnof phenyl-Sepharose [variants SakSTAR(E35A,E38A,R77A),SakSTAR(E38A,E75A), SakSTAR(K35A,E75A,R77A), SakSTAR(K35A,E75A),SakSTAR(K74A), SakSTAR(E75A), SakSTAR(E80A), SakSTAR(D82A) andSakSTAR(E75A,D82A)]. The SakSTAR containing fractions, localized bySDS-gel electrophoresis, were pooled for further analysis.

Physicochemical and Biochemical Analysis

Protein concentrations were determined according to Bradford (29). Thespecific activities of SakSTAR solutions were determined with achromogenic substrate assay carried out in microtiter plates using amixture of 80 μL SakSTAR solution and 100 μL Glu-plasminogen solutionprepared as described elsewhere (30) (final concentration 0.5 μmol/L).After incubation for 30 min at 37° C., generated plasmin was quantitatedby addition of 20 μL S2403 (final concentration 1 mmol/L) andmeasurement of the absorption at 405 nm. The activity was expressed inhome units (HU) by comparison with an in-house standard (lot STAN5)which was assigned an activity of 100,000 HU (100 kHU) per mg, proteinas determined by amino acid composition (7). SDS-PAGE was performed withthe Phast System™ (Pharmacia, Uppsala, Sweden) using, 10-15% gradientgels and Coomassie Brilliant blue staining. Reduction of the samples wasperformed by heating at 100° C. for 3 min in the presence of 1% SDS and1% dithioerythritol. The specific activities of the different SakSTARmutants determined with the chromogenic substrate assay are summarizedin Table 1.

Binding to Murine Monoclonal Antibodies

In agreement with previous observations (22), SakSTAR(K74A,E75A,R77A)did not react with 4 of the 5 MAbs recognizing epitope I, whereasSakSTAR(K35A,E38A) did not react with 3 of the 5 and SakSTAR(E80A,D82A)not with 4 of the 5 Mabs recognizing epitope III. These reducedreactivities were additive in SakSTAR(K35A,E38A,K74A,E75A,R77A) and inSakSTAR(K74A,E75A,R77A,E80A,D82A). The reduced reactivity ofSakSTAR(K74A,E75A,R77A) was fully maintained inSakSTAR(K35A,E38A,K74A,E75A) and in SakSTAR(K35A,E75A,R77A), largely inSakSTAR(K35A,E38A,E75A,R77A), SakSTAR(E38A,E75A,R77A),SakSTAR(E38A,E75A) and SakSTAR(E75A), but much less inSakSTAR(K35A,E38A,K74A,R77A) and SakSTAR(K74A), indicating that E75 isthe main contributor to the binding of the 4 Mabs recognizing epitope Iof SakSTAR. However, surprisingly, binding of epitope I antibodies toSakSTAR(E75A,D82A) was normal in two independent preparations fromexpression plasmids with confirmed DNA sequences. The reduced reactivityof the 3 MAbs of epitope III with SakSTAR(K35A,E38A) required both K35and E38, as demonstrated with SakSTAR(E38A,K74A,E75A,R77A) andSakSTAR(K35A,K74A,E75A,R77A), with SakSTAR(E38A,E75A) and SakSTAR(K35A,E75A) and with SakSTAR(E38A,E75A,R77A) and SakSTAR(K35A,E75A,R77A). Thereduced reactivity of the 4 MAbs of cluster mI with SakSTAR(E80A,D82A)was maintained in SakSTAR(D82A) but not in SakSTAR(E80A).

Absorption of Antibodies, Elicited in Patients by Treatment withWild-type SakSTAR

Plasma samples from 16 patients with acute myocardial infarction,obtained several weeks after treatment with SakSTAR (4,31) were used.The staphylokinase-neutralizing activity in these samples was determinedas follows. Increasing concentrations of wild-type or variant SakSTAR(50 μL volumes containing 0.2 to 1000 μg/mL) were added to a mixture of300 μL citrated human plasma and 50 μL buffer or test plasma,immediately followed by addition of 100 μL of a mixture containingthrombin (50 NIH units/mL) and CaCl₂ (25 mmol/L). The plasma clot lysistime was measured and plotted against the concentration of SakSTARmoiety. From this curve the concentration of staphylokinase moiety thatproduced complete clot lysis in 20 min was determined. The neutralizingactivity titer was determined as the difference between the test plasmaand buffer values and was expressed in μg per mL test plasma. Theresults of the individual patients have been reported elsewhere (22).For the present invention, three plasma pools were made, one from 10patients from whom sufficient residual plasma was available, one fromthree patients that absorbed less than 50% of the antibodies withSakSTAR(K35A,E38A,K74A,E75A,R77A) (Subpool B) and one from threepatients that absorbed >90% of the antibodies withSakSTAR(K35A,E38A,K74A,E75A, R77A) (Subpool C).

These plasma pools were diluted (1/30 to 1/200) until their binding toSakSTAR substituted chips in the BIAcore instrument amounted toapproximately 2000 RU. From this dilution a calibration curve forantibody binding was constructed using further serial two-folddilutions. The plasma pools were absorbed for 10 min with 100 nmol/L ofthe SakSTAR variants, and residual binding to immobilized SakSTAR wasdetermined. Residual binding was expressed in percent of unabsorbedplasma, using the calibration curve.

The results are summarized in Table 1. Whereas wild-type SakSTARabsorbed more than 95% of the binding antibodies from pooled plasma ofthe 10 patients, incomplete absorption (<60%) was observed withSakSTAR(K74A,E75A,R77A), SakSTAR(K35A,E38A,K74A, E75A,R77A),SakSTAR(E38A,K74A,E75A,R77A), SakSTAR(K35A,K74A,E75A,R77A),SakSTAR(K35A,E38A,K74A,R77A), SakSTAR(K35A,E38A,K74A,E75A),SakSTAR(K74A) and SakSTAR(K74A,E75A,R77A,E80A,D82A) but absorption wasnearly complete with SakSTAR(K35A,E38A), SakSTAR(K35A,E38A,E75A,R77A),SakSTAR(E38A,E75A,R77A), SakSTAR(E38A,E75A), SakSTAR(K35A,E75A,R77A),SakSTAR(K35A,E75A), SakSTAR(E75A), SakSTAR(E80A,D82A), SakSTAR(E80A),SakSTAR(D82A) and SakSTAR(E75A,D82A). These results, surprisingly,demonstrate that approximately 40% of the antibodies elicited inpatients by treatment with wild-type SakSTAR depend on K74 for theirbinding (Table 1). Absorption with pooled plasma from 3 patients fromwhich <50% of the antibodies were absorbed withSakSTAR(K35A,E38A,K74A,E75A,R77A) (Subpool B) confirmed the predominantrole of K74 for antibody recognition. As expected, absorption withpooled plasma from 3 patients from which >95% of the antibodies wereabsorbed with SakSTAR(K35A,E38A,K74A,E75A,R77A) (Subpool C) was nearlycomplete with all variants tested.

EXAMPLE 3

Comparative Thrombolytic Efficacy and Immunogenicity ofSakSTAR(K74A,E75A,R77A) and SakSTAR(K74A) Versus SakSTAR in Patientswith Peripheral Arterial Occlusion

Purification of SakSTAR(K74A,E75A,R77A) and SakSTAR(K74A) for Use invivo

A 12 to 24 L culture (in 2 L batches) of the variantsSakSTAR(K74A,E75A,R77A), or of SakSTAR(K74A) was grown and IPTG-inducedin LB medium supplemented with 100 μg/mL ampicillin, pelleted,resuspended, disrupted by sonication and cleared as described above. Thecompounds were purified by chromatography on a 5×20 cm column ofSP-Sephadex, a 5×10 cm column of Q-Sepharose and/or a 5×13 cm column ofphenyl-Sepharose using buffer systems described elsewhere (22,23). Thematerials were then gel filtered on sterilized Superdex 75 to furtherreduce their endotoxin content. The SakSTAR variant containing fractionswere pooled, the protein concentration was adjusted to 1 mg,/mL and thematerial sterilized by filtration through a 0.22 μm lylillipore filter.The methodology used to determine the biological properties of the finalmaterial required for use in vivo is described above and elsewhere (22).

Materials and Methods

Staphylokinase-neutralizing activity in plasma was determined asdescribed above. Quantitation of antigen-specific IgG and IgM antibodieswas performed using enzyme-linked immunosorbent assays in polystyrenemicrotiter plates essentialy as described previously (22). In the IgGassays, dilution curves of affinospecific anti-SakSTAR IgG antibodieswere included on each plate. These antibodies were isolated from plasmaobtained from 3 patients, after thrombolytic therapy with wild-typeSakSTAR, by chromatography on protein A-Sepharose and on insolubilizedSakSTAR, and elution of bound antibodies with 0.1 mol/L glycine-HCl, pH2.8. The purity of the IgG preparation was confirmed by sodiumdodecylsulfate polyacrylarnide gel electrophoresis. In the IgM assays,titers defined as the plasma dilution giving an absorbancy at 492 nmequivalent to that of a 1/640 dilution of pooled plasma were determinedand compared with the titer of baseline samples before treatment (medianvalue 1/410, interquartile range 1/120-1/700).

Thrombolytic Efficacy

Wild-type SakSTAR or the variants SakSTAR(K74A) orSakSTAR(K74A,E75A,R77A) were administered intra-arterially at or in theproximal end of the occlusive thrombus as a bolus of 2 mg followed by aninfusion of 1 mg/hr (reduced overnight in some patients to 0.5 mg/hr) ingroups of 6 to 12 patients with angiographically documented occlusion ofa peripheral artery or bypass graft of less than 120 days duration.Patients were studied after giving informed consent, and the protocolwas approved by the Human Studies Committee of the University of Leuven.Inclusion and exclusion criteria, conjunctive antithrombotic treatment(including continuous intravenous heparin) and the study protocol wereessentially as previously described (22).

Relevant baseline characteristics of the individual patients are shownin Table 2. The majority of PAO were at the femoropopliteal level. Twoiliac stent and 8 graft occlusions were included. Eight patientspresented with incapacitating claudication, 5 with chronic ischemic restpain, 7 with subacute ischemia and 7 with acute ischemia. One patient(POE) who had 2 years previously been treated with SakSTAR was includedin the SakSTAR(K74A) group. This patient was not included in thestatistical analyses.

Table 2 also summarizes the individual treatment and outcome.Intra-arterial infusion, at a dose of 6.0 to 25 mg and a duration of 4.0to 23 hrs, induced complete recanalization in 24 patients and partialrecanalization in 3. Complementary endovascular procedures (mainly PTA)were performed in 17 patients and complementary reconstructive vascularsurgery following thrombolysis in 3. No patient underwent majoramputation. Early recurrence of thrombosis after the end of theangiographic procedure occurred in 4 patients. Bleeding complicationswere absent or limited to mild to moderate hematoma formation at theangiographic puncture sites except for 5 patients who requiredtransfusion (data not shown). Intracranial or visceral hemorrhage wasnot observed. Circulating, fibrinogen, plasminogen and α₂-antiplasminlevels remained essentially unchanged during infusion of the SakSTARmoieties (data not shown), confirming absolute fibrin specificity ofstaphylokinase at the dosages used. Significant in vivo fibrin digestionoccurred as evidenced by elevation of fibrin fragment D-dimer levels.Intra-arterial heparin therapy prolonged aPTT levels to a variableextent (data not shown).

Antibody Induction

Antibody-related SakSTAR-, SakSTAR(K74A)- andSakSTAR(K74A,E75A,R77A)-neutralizing, activity and anti-SakSTAR,anti-SakSTAR(K74A) and anti-SakSTAR(K74A,E75A,R77A) IgG, were low atbaseline and during the first week after the infusion (FIG. 2). From thesecond week on, neutralizing activity levels increased to reach medianvalues at 3 to 4 weeks of 20 μg SakSTAR(K74A) and 2.4 μgSakSTAR(K74A,E75A,R77A) neutralized per mL plasma in the patientstreated with SakSTAR(K74A) and SakSTAR(K74A,E75A,R77A), respectively,which is significantly lower than the median value of 93 μg wild-typeSakSTAR neutralized per mL in the patients treated with SakSTAR (p=0.024for differences between the three groups by Kruskal-Wallis analysis andp=0.01 and p=0.036, respectively, for variants vs wild-type byMann-Whitney rank sum test). The levels of anti-SakSTAR(K74A) and ofanti-SakSTAR(K74A,E75A,R77A) IgG increased to median values at 3 to 4weeks of 270 and 82 μg/mL plasma in patients treated with SakSTAR(K74A)and SakSTAR(K74A,E75A,R77A) respectively, which is significantly lowerthan the median value of 1800 μg anti-SakSTAR per mL plasma in thepatients treated with SakSTAR ((p=0.024 for differences between thethree groups by Kruskal-Wallis analysis and p=0.007 and 0.05,respectively, for variants versus wild-type by Mann-Whitney rank sumtest).

The titers of anti-SakSTAR(K74A) and of anti-SakSTAR(K74A,E75A,R77A) IgMincreased from median baseline values of 1/460 and 1/410 to medianvalues at 1 week of 1/510 and 1/450 in patients treated withSakSTAR(K74A) and SakSTAR(K74A,E75A,R77A), respectively, which was notsignificantly different from the median values of 1/320 at baseline and1/640 at week 1 in patients treated with SakSTAR. Corresponding valuesat 2 weeks were 1/590 and 1550 in patients given SakSTAR(K74A) andSakSTAR(K74A,E75A,R77A), not significantly different from 1/930 withSakSTAR (data not shown).

The antibodies induced by treatment with SakSTAR were completelyabsorbed by SakSTAR but incompletely by SakSTAR(K74A) and bySakSTAR(K74A,E75A,R77A) confirming the immunogenicity of the K74,E75,R77epitope and the dominant role of K74 in the binding of antibodiesdirected against this epitope. The antibodies induced by treatment withSakSTAR(K74A) or SakSTAR(K74A,E75A,R77A) were completely absorbed bySakSTAR, by SakSTAR(K74A) and by SakSTAR(K74A,E75A,R77A), indicatingthat immunization was not due to neoepitopes generated by substitutionof Lys74 with Ala, but to epitopes different from the K74,E75,R77epitope.

Thus, this example illustrates that staphylokinase variants with reducedantibody induction but intact thrombolytic potency can be generated. Thepresent experience in 26 patients treated with SakSTAR (n=9),SakSTAR(K74A) (n=11) and SakSTAR(K74A,E75A,R77A) (n=6) combined withprevious experience in 14 patients with SakSTAR (n=7) andSakSTAR(K35A,E38A,K74A,E75A,R77A) (n=7) (31) and in 24 patients withSakSTAR (32), and with subsequent non-randomized experience in patientswith SakSTAR (n=30) with SakSTAR(K74A) (n=12) and withSakSTAR(K74A,E75A,R77A) (n=7) (data not shown), allows an initialestimation of the prevalence of immunization by intra-arterial treatmentwith SakSTAR or variants with an altered K74,E75,R77 epitope[SakSTAR(K74A), SakSTAR(K74A,E75A,R77A) and SakSTAR(K35A,E38A,K74A,E75A,R77A)]. Neutralizing activity data after 2 to 4 weeks, available in 70patients with peripheral arterial occlusion given intra-arterialSakSTAR, revealed that 56 patients (80 percent) had levels >5 μgcompound neutralized per mL plasma. Of the patients given SakSTAR(K74A),SakSTAR(K74A,E75A,R77A) or SakSTAR(K74A,E75A,K74A,E75A,R77A), 27 of the43 (63 percent) had neutralizing activity levels of >5 μg compound permL plasma. This difference is statistically significant (p=0.05 byFisher's exact test) indicating that the K74,E75,R77 epitope is a majordeterminant of antibody induction. However, the residual prevalence ofspecific immunocompetence against SakSTAR(K74A) indicates thatadditional mutagenesis to further reduce the immunogenicity of SakSTARvariants for clinical use, would be desirable.

EXAMPLE 4

Construction, Epitope Mapping with Murine Monoclonal Antibodies andAbsorption with Pooled Plasma of Immunized Patients, ofAlanine-substitution Mutants of Staphylokinase

Site-directed mutagenesis was applied to residues other than “chargedamino acids” in order to identify i) additional residues belonging toepitopes I and III identified with the panel of murine Mabs and ii)amino acids determining absorption to antiserum from immunized patients.Since functional epitopes generally comprise more than one amino acidresidue critical for antibody binding, identification of additionalresidues in these epitopes could lead to the construction of newcombination derivatives displaying a lower antigenic profile, whilekeeping the specific activity and the temperature stability of wild-typestaphylokinase.

In this example, the construction and characterization of SakSTARvariants in which one or at most two amino acids (adjacent or in closevicinity) were substituted with alanine is described. The mutantsdescribed under this example are listed in Table 3. These variants wereexpressed in E. coli, purified and characterized in terms of specificactivity, reactivity with the panel of murine monoclonal antibodies, andabsorption of antibodies from plasma of patients treated with wild-typeSakSTAR.

Reagents and Methods

The source of all reagents used in the present study has previously beenreported (22), or is specified below. The template vector formutagenesis, pMEX602sakB (i.e. pMEX.SakSTAR), has been describedelsewhere (23). Restriction and modification enzymes were purchased fromNew England Biolabs (Leusden, The Netherlands), Boehringer Mannheim(Mannheim, Germany) or Pharmacia (Uppsala, Sweden). The enzymaticreactions were performed according to the supplier recommendation. Themutagenic oligonucleotides and primers were obtained from Eurogentec(Seraing, Belgium). Plasmid DNA was isolated using a purification kitfrom Qiagen (Hilden, Germany) or the BIO 101 RPM kit (Vista, Calif.), asrecommended. Transformation-competent E. coli cells were prepared by thewell-known calcium phosphate procedure. Nucleotide sequencedetermination was performed on double strand plasmid DNA with thedideoxy chain termination method, using the T7 sequencing kit(Pharmacia, Uppsala, Sweden). Polymerase chain reactions (PCR) wereperformed using Taq polymerase from Boehringer Mannheim (Mannheim,Germany) or Vent polymerase (New England Biolabs, Leusden, TheNetherlands). The recombinant DNA methods required to construct thevariants described in this example are well established (22,27).

Construction of Expression Plasmids

The variants SakSTAR(Y17A,F18A), SakSTAR(F104A), SakSTAR(F111A),SakSTAR(Y9A), SakSTAR(Y91A), SakSTAR(Y92A), SakSTAR(I87A),SakSTAR(I106A) and SakSTAR(I120A) were constructed with the ChameleonDouble-Stranded Site-Directed Mutagenesis kit from Stratagene (La Jolla,USA), using the pMEX.SakSTAR vector as template, and followinginstructions of the supplier. The mutagenic oligonucleotides (not shown)were used in combination with the selection-primer LY34 5′CAAAACAGCCGAGCTTCATTCATTCAGC (SEQ ID NO:3), which destroys the uniqueHindIII site located 3′ to the staphylokinase encoding gene inpMEX.SakSTAR and allows to counter-select the non-mutant progeny byHindIII digestion. The deletion of the HindIII site was in most casescorrelated with the presence of the desired mutation introduced by themutagenic oligonucleotide. The variant SakSTAR(I133A), was constructedby perforning a polymerase chain reaction on the pMEX.SakSTAR plasmidusing the primer 818A located at the 5′ end of the sakSTAR gene (5′CAGGAAACAGAATTCAGGAG) (SEQ ID NO:1) and the mutagenic primer LY58 (5′TTCAGCATGCTGCAGTTATTTCTTTTCTGCAACAACCTTGG) (SEQ ID NO:4). The amplifiedproduct (30 cycles: 30 sec at 94° C., 30 sec at 50° C., 30 sec at 72°C.) was purified, digested with EcoRI and PstI, and ligated into thecorresponding sites of pMEXSakSTAR.

The variants SakSTAR(I128A), SakSTAR(L127A) and SakSTAR(N126V) wereconstructed by performing a polymerase chain reaction using the primer818A located at the 5′ end of the sakSTAR gene and mutagenic primers(not shown). The amplified product (30 cycles: 1 sec at 94° C., 1 sec at50° C., 10 sec at 72° C.) was purified, digested with EcoRI and StyI,and ligated into the corresponding sites of pMEXSakSTAR. The variantSakSTAR(F125A) was constructed by performing two consecutive PCRreactions (30 cycles: : 30 sec at 94° C., 30 sec at 50° C., 30 sec at72° C.). In the first reaction, a fragment of pMEX.SakSTAR was amplifiedwith the primers 818A and a mutagenic primer. This amplified fragmentwas then used as template in a second PCR reaction with a mutagenicprimer in order to further elongate the fragment downstream of the StyIsite present in the sakSTAR gene (corresponding to amino acids 130-131of SakSTAR). The resulting product was digested with EcoRI and StyI, andligated into the corresponding sites of pMEXSakSTAR.

The plasmids encoding all the other variants listed in Table 3 wereconstructed by direct PCR or by the spliced overlap extension polymerasechain reaction (SOE-PCR)(24) using pMEX.SakSTAR or available plasmnidsencoding SakSTAR variants as template. Two fragments were amplified byPCR (30 cycles: 1 sec at 94° C, 1 sec at 50° C., 10 sec at 72° C.), thefirst one starting from the 5′ end (primer 818A ) of the staphylokinasegene to the region to be mutagenized (forward primer), the second onefrom this same region (backward primer) to the 3′ end of the gene withprimer 818D (5′ CAAACAGCCAAGCTTCATTCATTCAGC) (SEQ ID NO:5). The forwardand backward primers shared an overlap of around 24 bp (primers notshown). The two purified fragments were then assembled together in asecond PCR reaction with the external primers 818A and 818D (30 cycles:1 sec at 94° C., 1 sec at 50° C., 10 sec at 72° C.). The amplifiedproduct from this final reaction was purified, digested with EcoRI andHindIII and ligated into the corresponding site of pMEX.SakSTAR. Foreach construction, the sequence of the variant was confirmed bysequencing the entire SakSTAR coding region.

Expression and Purification of SakSTAR Variants

The SakSTAR variants were expressed and purified, as described below,from transformed E. coli grown in terrific broth (TB) medium (28). A 2to 4 mL aliquot of an overnight saturated culture in LB medium was usedto inoculate a 1 to 2 L culture in terrific broth supplemented with 100μg/mL ampicillin. The culture was incubated with vigorous aeration andat 30° C. After about 16 hours incubation, IPTG (200 μmol/L) was addedto the culture to induce expression from the tac promoter. After 3 hoursinduction, the cells were pelleted by centrifugation at 4,000 rpm for 20min, resuspended in 1/10 volume of 0.01 mol/L phosphate buffer pH 6-6.5and disrupted by sonication at 0° C. The suspension was centrifuged for20 min at 20,000 rpm and the supematant was stored at 4° C. or at −20°C. until purification. The material was purified essentially asdescribed above (Example 2): cleared cell lysates containing the SakSTARvariants were subjected to chromatography on a 1.6×5 cm column ofSP-Sephadex, followed by chromatography on a 1.6×8 cm column ofphenyl-Sepharose. The SakSTAR containing fractions, localized by SDS-gelelectrophoresis, were pooled for further analysis.

Physicochemical and Biochemical Analysis

Protein concentrations were determined according to Bradford (29).SDS-PAGE was performed with the Phast System™ (Pharmacia, Uppsala,Sweden) using 10-15% gradient gels and Coomassie Brillant blue staining,and the specific activities of SakSTAR solutions were determined with achromogenic substrate assay carried out in microtiter plates (asdescribed in example 2). The specific activity of the different SakSTARvariants are summarized in Table 3.

Reactivity of SakSTAR Variants with a Panel of Murine MonoclonalAntibodies

The methodology used to determine the reactivity of the SakSTAR variantswith a panel of murine monoclonal antibodies was described in example 1above. The results are summarized in Table 3 (the layout of this Tablecorresponds to the layout of Table 1, as described in example 1).Apparent association constants at least 10-fold lower than those ofwild-type staphylokinase were considered as significant and areindicated in bold type in the table.

In order to obtain a comprehensive inventory of the properties ofAla-substitution variants of the SakSTAR molecule, 67 plasmids encodingvariants with substitution of a single or two adjacent amino acids withAla were constructed, expressed and purified. Together with the 35charged residue to Ala-substitution variants previously described (22,and example 2), this analysis covers all residues in SakSTAR except Gly,Ala and Pro, as illustrated in FIG. 3. Eight of the variants could notbe obtained in purified form, primarily as a result of low expressionlevels, 11 variants were inactive, 56 had a reduced specific activity,and 27 had a maintained or increased specific activity (≧100 kHU/mg).The yields of purified material from cultures of expressed plasmids were16 mg/L (median, 10 to 90 percentile range 4 to 41 mg/L). SDSpolyacrylamide gel electrophoresis consistently showed one main bandwith M_(r)≈16,000, usually representing ≧95% of total protein (notshown).

Substitution of K35, N95, S103 or K135 with Ala yielded variants withspecific activities of ≧200 kU/mg. Substitution of W66, Y73 or E75 withAla reduced the reactivity of the variants with ≧3 antibodies of epitopecluster I, of H43 or V45 with Ala that with 3 antibodies from epitopecluster II and of V32, K35, D82 and K130 with Ala that with ≧3antibodies of epitope cluster III.

Absorption of Antibodies, Elicited in Patients by Treatment with SakSTAR

For the present example, the three plasma pools, as described in example2 were used. The methodology used to evaluate the absorption withwild-type staphylokinase and with SakSTAR variants, of antibodieselicited in patients treated with SakSTAR, is described in detail inexample 2. The results are summnarized in Table 3. Whereas wild-typeSakSTAR and most of the variants analyzed in this example absorbed morethan 95% of the binding antibodies from pooled plasma of the 10patients, incomplete absorption (<60%) was observed with SakSTAR(Y73A),and with SakSTAR(K74A). The predominant role of Lys74 for antibodyrecognition has been demonstrated previously (see example 2). Thepresent results indicate that Tyr73 participates to the same majorepitope as Lys74, or, alternatively, that substitution at Tyr73 mayindirectly induce a structural modification of the “K74-epitope”.Absorption with pooled plasma from 3 patients from which >95% of theantibodies were absorbed with SakSTAR(K35A,E38A,K74A,E75A,R77A) (SubpoolC, see example 2) was nearly complete with most variants tested.

EXAMPLE 5

Construction, Epitope Mapping with Murine Monoclonal Antibodies andAbsorption with Pooled Plasma of Immunized Patients, of StaphylokinaseVariants with Substitution of S34, G36 and/or H43

The natural variant Sak42D differs from SakSTAR in three amino acids andcorresponds to SakSTAR(S34G,G36R,H43R). Sak42D is characterized byreduced reactivity with some murine antibodies of epitope clusters IIand III and a slightly reduced absorption of antibodies from plasma ofpatients treated with SakSTAR (Table 4). Mutagenesis of these residuesin SakSTAR revealed that the reduced reactivity with epitope cluster Inand with immunized patient plasma could be ascribed to the G36Rsubstitution, the H43R substitution mediated the reduced reactivity withepitope cluster II but had no effect on the reactivity with immunizedpatient plasma, whereas the S34A substitution had no effect. The G36Rsubstitution could be combined with the K74R but not with the K74Asubstitution, without significant reduction of the specific activity(Table 4).

EXAMPLE 6

Construction, Epitope Mapping with Murine Monoclonal Antibodies andAbsorption with Pooled Plasma of Immunized Patients, of StaphylokinaseVariants with Substitution of K35, E65, Y73, K74, E80+D82, N95, K130,V132 and/or K135

Based on the results of the alanine-substitution analysis in example 4,K35, N95 and K135 were selected for further analysis becauseSakSTAR(K35A), SakSTAR(N95A) and SakSTAR(K135A) had a two-fold increasedspecific activity, Y73 and K74 because SakSTAR(Y73A) and SakSTAR(K74A)had a markedly reduced reactivity with antibodies from epitope cluster Iand diminished absorption of antibodies from plasma of patientsimmunized by treatment with SakSTAR, and K35, E80+D82, K130 and V132because SakSTAR(K35A), SakSTAR(E80A,D82A), SakSTAR(K130A) andSakSTAR(V132A) had a reduced reactivity with antibodies from epitopecluster III.

In an effort to maximize the activity/antigenicity ratio, these aminoacids were substituted with other amino acids than Ala. As summarized inTable 5, substitution of K35 with A, E or Q revealed that SakSTAR(K35A)had the most interesting properties, substitution of Y73 with F, H, L, Sor W did not rescue the marked reduction in specific activity, and K74confirmed its key role in binding of antibodies from immunized patientplasma, the best specific activity/antigenicity ratios being obtainedwith SakSTAR(K74Q) and SakSTAR(K74R). SakSTAR(E80A,D82A) was preferredover the single residue variants SakSTAR(E80A) or SakSTAR(D82A) becauseof its somewhat lower reactivity with immunized patient plasma.SakSTAR(N95A) could not be further improved by substitution of N95 withE, G, K or R and it was unable to confer its increased specific activityto variants containing K74A or K135R. Finally SakSTAR(K130A) wasoutperformed in terms of specific activity by SakSTAR(K130T) andSakSTAR(V132A) by SakSTAR(V132R).

EXAMPLE 7

Construction, Epitope Mapping with Murine Monoclonal Antibodies andAbsorption with Pooled Plasma of Immunized Patients of CombinationVariants of SakSTAR(K130T,K135R) and SakSTAR(E80A,D82A,K130T,K135R) withK35A,G36R,E65X,K74X and Selected Other Amino Acids

In the present and the following examples an additional plasma pool wasmade from 40 patients obtained several weeks after treatment withSakSTAR (Pool 40). The original pool from 10 patients is furtheridentified as Pool 10. The absorption of staphylokinase-specificantibodies was quantified as described above and elsewhere (22).

The SakSTAR(K130T,K135R) variant was taken as a template for additivemutagenesis because of its high specific activity with a moderatereduction of binding to antibodies of epitope cluster III and absorptionof antibodies from immunized patient plasma (Table 6). Addition of G36R,K74R, or K74Q or both to the template did not markedly reduce thespecific activity, reduced the reactivity with monoclonal antibodiesagainst epitope cluster III (G36R substitution) and decreased theabsorption of antibodies from immunized patient plasma (K74R or K74Qsubstitution). Combination of E65A or E65Q with K74Q in theSakSTAR(K130T,K135R) template reduced the absorption of antibodies fromPool 10 and Pool 40 to around 50 and 60 percent respectively, withoutmarkedly reducing the specific activity. Addition substitution ofselected amino acids in the SakSTAR(E65Q,K74Q,K130T,K135R) template didnot further reduce the antibody absorption from Pool 10 or Pool 40.Surprisingly, the substitution of K136 with A and the addition of K inposition 137 resulted in a marked increase in specific activity, asmeasured in the chromogenic substrate assay.

Combination of the SakSTAR(E80A,D82A) and SakSTAR,(K130T,K135R)templates, did not affect the specific activity and had a reducedreactivity with epitope cluster III antibodies (Table 7). Therefore theSakSTAR(E80A,D82A,K130T,K135R) template was selected for furthermutagenesis. Addition of K74R and even more of K74Q drastically reducedthe reactivity with immunized patient plasma. Finally, addition of E65Dor of K35A or E65S to the SakSTAR(K74R,E80A,D82A,K130T,K135R) orSakSTAR(K74Q,E80A,D82A,K130T, K135R) templates yielded variants withintact specific activity which only bound ≦45 of the antibodies ofpooled immunized patient plasma and less than 15 percent of the subpoolreacting for more than 50 percent with the K74,E75,R77 epitope.

EXAMPLE 8

Characterization of Selected Variants of Staphylokinase with IntactSpecific Activity and Less Than 50% Adsorption of Pooled SakSTARSpecific Human Antibodies Elicited in Patients by Treatment withWild-type SakSTAR

Twenty three of the variants constructed and characterized in the aboveexamples combined the properties of a residual specific activity of ≧100kHU/mg and ≦50 percent absorption with the pool of antisera obtainedfrom 10 patients treated with wild-type SakSTAR. The results aresummarized in Table 8. Results obtained with Subpool B and Subpool C andwith the pool of 40 patients treated with wild-type SakSTAR areincluded. SakSTAR(K74Q,E80A,D82A,K130T,K135R),SakSTAR(E65D,K74R,E80A,D82A,K130T, K135R),SakSTAR(K35A,E65D,K74Q,E80A,D82A,K130T,K135R) and SakSTAR(E65Q,K74Q,N95A,E118A,K130A,K135R,K136A,+137K) were selected for furthercharacterization.

Fibrinolytic Properties of SakSTAR Variants in Human Plasma in vitro

The fibrinolytic and fibrinogenolytic properties of the SakSTAR variantswere determined as previously described. Dose- and time-dependent lysisof ¹²⁵I-fibrin labeled human plasma clots submerged in human plasma wasobtained with the selected variants (Table 9). Spontaneous clot lysisduring the experimental period was ≦5% (not shown). Equi-effectiveconcentrations of test compound (causing 50% clot lysis in 2 hrs; C₅₀),determined graphically from plots of clot lysis at 2 hrs versus theconcentration of plasminogen activator (not shown), ranged from0.11±0.01 to 0.24±0.04 μg/mL at which the residual fibrinogen levelsranges between 92±30 and 97±30 percent of baseline (Table 9). Theconcentrations of compound causing 50% fibrinogen degradation in 2 hrsin human plasma in the absence of fibrin were determined graphicallyfrom dose-response curves (not shown). These values (mean±SD of 3independent experiments) ranged from 14±3.2 to 29±3.1 μg/mL (Table 9).Surprisingly the very high specific activity ofSakSTAR(E65Q,K74Q,N95A,E118A, K130A,K135R,K136A,+137K) in thechromogenic assay was not associated with an increased thrombolyticpotency in a plasma milieu.

Temperature Stability of Selected SakSTAR Variants

The temperature stability of preparations ofSakSTAR(K74Q,E80A,D82A,K130T,K135R), SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) and SakSTAR(K35A,E65D,K74Q, E80A,D82A,K130T,K135R),dissolved to a concentration of 1.0 mg/mL in 0.15 mol/L NaCl, 0.01 mol/Lphosphate buffer, pH 7.5 at various temperatures is illustrated in FIG.4. At temperatures up to 37° C., all compounds remained fully active forup at least three days. At 56° C. and 70° C. the three variants werehowever less stable than wild-type SakSTAR.

Pharmacokinetic Properties of SakSTAR Variants Following Bolus Injectionin Hamsters

The pharmacokinetic parameters of the disposition of SakSTAR variantsfrom blood were evaluated in groups of 4 hamsters following intravenousbolus injection of 100 μg/kg SakSTAR variant. SakSTAR-related antigenwas assayed using the ELISA described elsewhere. The ELISA wascalibrated against each of the SakSTAR variants to be quantitated.Pharmacokinetic parameters included: initial half-life (in min),t1/2α=ln2/α; terminal half-life (in min), t1/2β=ln2/β; volume of thecentral (plasma) compartment (in mL), V_(C)=dose/(A+B); area under thecurve (in μg.min.mL⁻¹), AUC=A/α+B/β; and plasma clearance (in mL.min⁻¹),Clp=dose/AUC (33).

The disposition rate of staphylokinase-related antigen from bloodfollowing bolus injection of 100 μg/kg of the selected SakSTAR variantsin groups of 4 hamsters could adequately be described by a sum of twoexponential terms by graphical curve peeling (results not shown), fromwhich the pharmacokinetic parameters summarized in Table 10 werederived. The pharmacokinetic parameters of the mutants were not markedlydifferent from those of wild type SakSTAR. Initial plasma half-lives(t1/2(α)) ranged between 2.0 and 3.2 min and plasma clearances (Clp)between 1.6 and 4.1 mL/min.

EXAMPLE 9

Comparative Thrombolytic Efficacy and Immunogenicity of SakSTAR(K74Q,E80A,D82A,K130T,K135R) and SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R)Versus SakSTAR in Patients with Peripheral Arterial Occlusion

Purification for Use in vivo

Eighteen liter cultures (in 2 L batches) of the variantsSakSTAR(K74Q,E80A,D82A,K130T, K135R) and SakSTAR(E65D,K74R,ESOA,D82A,K130T,K135R) were grown for 20 hours in terrific broth medium (28),supplemented with 100 μg/mL ampicillin and induced with IPTG during thelast 3 hours. The cells were pelleted, resuspended in 1/10 volume of0.01 mol/L phosphate buffer, pH 6.0, disrupted by sonication and clearedby centrifugation. The compounds were purified by chromatography on a10×7 cm column of SP-Sepharose, equilibrated with 0.01 mol/L phosphatebuffer, pH 6.0 and eluted with a 1 mol/L NaCl gradient (3 columnvolumes). The fractions containing SakSTAR variant were pooled, solidNaCl was added to a concentration of 2.5 mol/L and the material waschromatographed on a 10×20 cm column of phenyl-Sepharose followed bystepwise elution with 0.01 mol/L phosphate buffer, pH 6.0. The materialswere desalted on a 10×45 cm column of Sephadex G25, concentrated byapplication on a 5×10 cm column of SP-Sepharose with stepwise elutionwith 1.0 mol/L NaCl and finally gel filtered on a 6×60 cm column ofSuperdex 75 equilibrated with 0.15 m NaCl, 0.01 mol/L phosphate buffer,pH 7.5 to further reduce their endotoxin content. The SakSTAR variantcontaining fractions were pooled, the protein concentration was adjustedto 1 mg/mL and the material sterilized by filtration through a 0.22 μmMillipore filter. The methodology used to determine specific activity,endotoxin contamination, bacterial sterility and toxicity in mice isdescribed above and elsewhere (22). The purity of the preparation wasevaluated by SDS gel electrophoresis on 10% gels to which 40 μg ofcompound was applied.

Out of culture volumes of 18 liters of SakSTAR variant, 840 mg ofSakSTAR(K74Q,E80A,D82A,K130T,K135R) with a specific activity of 140kHU/mg and 800 mg SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) with aspecific activity of 150 were purified. The endotoxin content was <0.1and 0.26 IU/mg. Gel filtration on HPLC revealed a single mainsymmetrical peak in the chromatographic range of the column,representing >98% of the eluted material (total area under the curve)(not shown). SDS gel electrophoresis of 40 μg samples revealed singlemain components (not shown). Preparations sterilized by filtrationproved to be sterile on 3 day testing as described elsewhere (22).Intravenous bolus injection of SakSTAR variants in groups of 5 mice (3mg/kg body weight), did not provoke any acute reaction, nor reducedweight gain within 8 days, in comparison with mice given an equal amountof saline (not shown).

Thrombolytic Efficacy

Wild-type SakSTAR or the variants SakSTAR(K74Q,E80A,D82A,K130T,K135R) orSakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) were administeredintra-arterially at or in the proximal end of the occlusive thrombus asa bolus of 2 mg followed by an infusion of 1 mg/hr (reduced overnight insome patients to 0.5 mg/hr) in groups of 15, 6 and 6 patientsrespectively with angiographically documented occlusion of a peripheralartery or bypass graft of less than 30 days duration. Patients werestudied after giving informed consent, and the protocol was approved bythe Human Studies Committee of the University of Leuven. Inclusion andexclusion criteria, conjunctive antithrombotic treatment (includingcontinuous intravenous heparin) and the study protocol were essentiallyas previously described (22).

Relevant baseline characteristics of the individual patients and resultsof treatment and outcome are shown in Table 11. Intra-arterial infusion,at a dose of 3.5 to 27 mg and a duration of 2 to 44 hrs, inducedcomplete recanalization in 22 patients and partial recanalization in 5.Complementary endovascular procedures (mainly PTA) were performed in 13patients and complementary reconstructive vascular surgery followingthrombolysis in 5. One patient underwent major amputation. Bleedingcomplications were usually absent or limited to mild to moderatehematoma formation at the angiographic puncture sites (data not shown).One patient, given wild-type SakSTAR suffered a non-fatal intracranialbleeding, one (BUE) a retroperitoneal hematoma and two (MAN and STRO) agastro-intestinal bleeding.

Circulating fibrinogen, plasminogen and a₂-antiplasmin levels remainedunchanged during infusion of the SakSTAR moieties (data not shown),reflecting absolute fibrin specificity of these agents at the dosagesused (data not shown). Significant in vivo fibrin digestion occurred asevidenced by elevation of fibrin fragment D-dimer levels. Intra-arterialheparin therapy prolonged aPTT levels to a variable extent (data notshown).

Antibody Induction

Staphylokinase-neutralizing activity in plasma and antigen-specific IgGantibodies were quantitated essentialy as described above and elsewhere(22).

Antibody-related SakSTAR-, SakSTAR(K74Q,E80A,D82A,K130T,K135R)- andSakSTAR(E65D,K74R,E80A,D82A,K130T,K135R)-neutralizing activity andanti-SakSTAR, anti-SakSTAR(K74Q,E80A,D82A,K130T,K135R) andanti-SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) IgG, were low at baselineand during the first week after the infusion (FIG. 5). From the secondweek on, neutralizing activity levels increased to reach median valuesat 3 to 4 weeks of 9 μg SakSTAR(K74Q, E80A,D82A,K130T,K135R) and 0.5 μgSakSTAR(E65D,K74R,E80A,D82A,K130T,K135) neutralized per mL plasma in thepatients treated with the corresponding moieties, respectively, ascompared to median value of 24 μg wild-type SakSTAR neutralized per mLin the 15 patients treated with SakSTAR. The levels of antiSakSTAR(K74Q,E80A, D82A,K130T,K135R) and of antiSakSTAR(E65D,K74R,E70A,K130T,K135R) IgG increased to median values at 3to 4 weeks of 420 and 30 μg/mL plasma in patients treated with thecorresponding moieties, respectively, as compared to a median value of590 μanti-SakSTAR per mL plasma in the patients treated with SakSTAR(FIG. 5). The prevalence of immunization, defined as neutralizingactivities in plasma after 2 to 4 weeks exceeding 5 μg/ml was 3 of 6patients (50 percent) with SakSTAR(K74Q,E80A,D82A,K130T,K135R), 1 of 6patients (17 percent) with SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R), ascompared to 56 of 70 patients (80 percent) with SakSTAR. This differenceis statistically highly significant (p=0.01 by 2×3 Chi square analysis).

The sntibodies induced by treatment with SakSTAR were completelyabsorbed by SakSTAR but incompletely bySakSTAR(K74Q,E80A,D82A,K130T,K135R) and bySakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) (Table 12). Antibodics inducedby treatment with SakSTAR(K74Q,E80A,D82A,K130T,K135R), detectable in 4of the 6 patients, were completely (≧90 percent) absorbed by SakSTAR, bySakSTAR(K74Q,E80A, D82A,K130T,K135R) and bySakSTAR(E65D,K74R,E80A,D82A,K130T,K135R), indicating that immunizationwas not due to neoepitopes generated by substitution of wild-type aminoacids. Antibodies induced by treatment withSakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) detectable in one patient (URB)were completely absorbed with SakSTAR(K74Q,E80A,D82A,K130T,K135R) andwith SakSTAR(E65D,K74Q,E80A,D82A,K130T,K135R) but incompletely (85%)with wild-type SakSTAR, suggesting that a small fraction of the inducedantibodies might be directed against a neoepitope in the variant usedfor infusion.

EXAMPLE 10

Construction, Purification and Characterization of Cysteine-substitutionMutants of Staphylokinase

Site-directed mutagenesis was applied to substitute exposed amino acidswith single cysteine residues in order to construct i) homodimeric formsof staphylokinase, upon formation of an intermolecular disulfide bridge,and ii) polyethylene glycol-conjugated molecules (PEG-derivatives). Theaim for this study is twofold: first, the clearance can be reduced byincreasing the size of the injected molecule (via dimerization orconjugation with large molecule such as PEG) and second, PEG-derivativeshave also been shown to induce a reduced immunoreactivity in animalmodels (for review, see ref. 34). In both cases, a prolonged half-lifein vivo could help to reduce the pharmacological dosis ofstraphylokinase in patients. This reduction could be accompanied with areduced immunogenic reaction against the thrombolytic agent.

In this example, the construction and characterization of two SakSTARvariants in which one single amino acid was substituted with cysteine isdescribed. The mutants described under this example are listed in Table13. These variants were expressed in E. coli, purified and characterizedto some extent in terms of specific activity, fibrinolytic properties inhuman plasma in vitro and pharmacokinetic properties following bolusinjection in hamsters.

Reagents and Methods

The source of all reagents used in the present study has previously beenreported (22), or is specified below. The template vector formutagenesis, pMEX602sakB (i.e. pMEX.SakSTAR), has been describedelsewhere (23). Restriction and modification enzymes were purchased fromNew England Biolabs (Leusden, The Netherlands), Boehringer Mannheim(Mannheim, Germany) or Pharmacia (Uppsala, Sweden). The enzymaticreactions were performed according to the supplier recommendation. Themutagenic oligonucleotides and primers were obtained from Eurogentec(Seraing, Belgium). Plasmid DNA was isolated using a purification kitfrom Qiagen (Hilden, Germany), as recommended. Transformation-competentE. coli cells were prepared by the well-known calcium phosphateprocedure. Nucleotide sequence determination was performed on doublestrand plasmid DNA with the dideoxy chain termination method, using theT7 sequencing kit (Pharmacia, Uppsala, Sweden). Polymerase chainreactions (PCR) were performed using Taq polymerase from BoehringerMannheim (Mannheim, Germany). The recombinant DNA methods required toconstruct the variants described in this example are well established(22,27).

Construction of Expression Plasmids

The variants SakSTAR(K102C) and SakSTAR(K109C), were constructed by thespliced overlap extension polymerase chain reaction (SOE-PCR) (24) usingpMEX.SakSTAR encoding SakSTAR as template. Two fragments were amplifiedby PCR (30 cycles: 1 sec at 94° C., 1 sec at 50° C., 10 sec at 72° C.),the first one starting from the 5′ end (primer 818A) of thestaphylokinase gene to the region to be mutagenized (forward primer),the second one from this same region (backward primer) to the 3′ end ofthe gene with primer 818D (5′ CAAACAGCCAAGCTTCATTCATTCAGC) (SEQ IDNO:5). The forward and backward primers shared an overlap of around 24bp (for the construction of K102C: TAT GAT AAG AAT TGC AAA AAA GAA GAA(backward) (SEQ ID NO:6) and TTC TTC TTT TTT GCA ATT CTT ATC ATA (SEQ IDNO:7) (forward), for the construction of K109C: AAA AAG AAG AAA CGT GCTCTT TCC CTA (backward) (SEQ ID NO:8) and TAG GGA AAG AGC ACO TTT CTT CTTTTT (forward) (SEQ ID NO:9). The two purified fragments were thenassembled together in a second PCR reaction with the external primers818A and 818D (30 cycles: 1 sec at 94° C., 1 sec at 50° C., 10 sec at72° C.). The amplified product from this final reaction was purified,digested with EcoRI and HindHIII and ligated into the corresponding siteof pMEX.SakSTAR. For each construction, the sequence of the variant wasconfirmed by sequencing the entire SakSTAR coding region.

Expression and Purification of SakSTAR Variants

The SakSTAR variants were expressed and purified, as described below,from transformed E. coli grown in terrific broth (TB) medium (28). A 2to 4 mL aliquot of an overnight saturated culture in LB medium was usedto inoculate a 1 to 2 L culture in terrific broth supplemented with 100μg/mL ampicillin. The culture was incubated with vigorous aeration andat 30° C. After about 16 hours incubation, IPTG (200 μmol/L) was addedto the culture to induce expression from the tac promoter. After 3 hoursinduction, the cells were pelleted by centrifugation at 4,000 rpm for 20min, resuspended in 1/10 volume of 0.01 mol/L phosphate buffer pH 6-6.5and disrupted by sonication at 0° C. The suspension was centrifuged for20 min at 20,000 rpm and the supernatant was stored at 4° C, or at −20°C. until purification. The material was purified essentially asdescribed above (Example 2): cleared cell lysates containing the SakSTARvariants were subjected to chromatography on a 1.6×5 cm column ofSP-Sephadex, followed by chromatography on a 1.6×8 cm column ofphenyl-Sepharose. The SakSTAR containing fractions, localized by SDS-gelelectrophoresis, were pooled for further analysis.

Biochemical Analysis

Protein concentrations were determined according to Bradford (29).SDS-PAGE was performed with the Phast System™ (Pharmacia, Uppsala,Sweden) using 10-15% gradient gels and Coomassie Brillant blue staining,and the specific activities of SakSTAR solutions were determined with achromogenic substrate assay carried out in microtiter plates (asdescribed in example 2). The specific activity of the different SakSTARvariants are summarized in Table 13.

Mutant SakSTAR(K102C) was essentially monomeric as visualized bySDS-PAGE and Coomassie Brillant blue staining. Its specific activity wascomparable to that of wild-type staphylokinase. In contrast,SakSTAR(K109C) showed a propensity to form dimers (>60%). This resultedin a markedly increased specific activity in the plasminogen-coupledchromogenic substrate assay (see Table 13). Upon reduction withdithiothreitol (DTT) (20-fold molar excess during 1.5 hour at 37° C.)and alkylation with iodoacetamide (100-fold molar excess during 1 hourat 37° C.), the K109C dimer is converted into a stable monomer and itsresulting specific activity is within the expected range towardswild-type staphylokinase (Table 13). This results confirms thatformation of homodimers is the unique determinant for this largeincrease in specific activity. Dimeric SakSTAR(K109C) was separated frommonomeric SakSTAR(K109C) by chromatography on Source S (Pharmacia)(5×50mm). Loading buffer was 10 mM phosphate, pH 6.0 and dimericSakSTAR(K109C) was eluted by a salt gradient (up to 1 M) in the samebuffer. The dimeric SakSTAR(K109C) (>95% pure) containing fractions,localized by SDS-gel electrophoresis, were pooled for further analysis.

Chemical Crosslinking of Cysteine Mutants of SakSTAR with PolyethyleneGlycol

The thiol group of the cysteine mutant SakSTAR(K102C) was targeted forcoupling with an activated polyethylene glycol, OPSS-PEG (ShearwaterPolymers Europe, Enschede, The Netherlands). OPSS-PEG is a 5 kDa PEGmolecule carrying a single activated thiol group at one end that reactspecifically at slightly alkaline pH with free thiols. Modification ofSakSTAR(K102C) was achieved by incubating the molecule (100 μM) with athree-fold excess of SS-PEG in a 5 mM phosphate, pH 7.9 solution at roomtemperature. The extent of the reaction was monitored by following therelease of 2-thiopyridone from OPSS-PEG at 412 nm. After reaction (about15 min), the excess of OPSS-PEG was removed by purifying the derivatizedSakSTAR(K102C-PEG) on a 1.6×5 cm column of SP-Sephadex as describedabove (see Example 2). The SakSTAR(K102C-PEG) containing fractions,localized by optical density at 280 nm, were pooled for furtheranalysis. SDS-PAGE analysis and Coomassie blue staining confirmed thatPEG crosslinking on SakSTAR(K102C) was quantitative. As shown in Table13, the specific activity of the PEG-derivative was only marginallyaffected when compared to that of wild-type staphylokinase.

Fibrinolytic Properties of SakSTAR Variants in Human Plasma in vitro

The fibrinolytic and fibrinogenolytic properties of SakSTAR variantswere determined as previously described. Dose- and time-dependent lysisof ¹²⁵I-fibrin labeled human plasma clots submerged in human plasma wasobtained with four molecules: SakSTAR(K109C) as dimer and as monomer(after reduction and alkylation with iodoacetamide), the monomericSakSTAR(K102C) and the PEG-derivatized SakSTAR(K102C). Spontaneous clotlysis during the experimental period was ≦5% (not shown). Equi-effectiveconcentrations of test compound (causing 50% clot lysis in 2 hrs; C₅₀),determined graphically from plots of clot lysis at 2 hrs versus theconcentration of plasminogen activator (not shown), were comparable tothat of SakSTAR, for monomeric SakSTAR(K109C) and SakSTAR(K102C) (Table13). However, it was observed that the C₅₀ for clot lysis by dimericSakSTAR(K109C) was only 0.12 μg/ml, which is approximately three foldlower than for wild-type staphylokinase. In contrast, a C₅₀ of 0.60μg/ml was measured for SakSTAR(K102C-PEG), which is only two-fold higherthan for wild-type staphylokinase. Thus, dimerization of SakSTAR viadisulfide bridges or increasing the size of the molecule viaPEG-derivatization does not preclude the fibrinolytic activity ofstaphlokinase. While a PEG-molecule appears to reduce the diffusion andtherefore fibrinolytic potency of the derivatized staphylokinase withina fibrin clot, dimerization of staphlokinase results in a synergisticfibrinolytic effect on human fibrin clots.

Pharmacokinetic Properties of Dimeric SakSTAR(K109C) andSakSTAR(K102C-PEG) Following Bolus Injection in Hamsters

The pharmacokinetic parameters of the disposition of dimericSakSTAR(K109C) and SakSTAR(K102C) from blood were evaluated in groups of4 hamsters following intravenous bolus injection of 100 μg/kg SakSTARvariant. SakSTAR-related antigen was assayed using the ELISA describedelsewhere. The ELISA was calibrated against each of the SakSTAR variantsto be quantitated. Pharmacokinetic parameters included: inital half-life(in min), t1/2α=ln2/α; terminal half-life (in min), t1/2β=ln2/β; volumeof the central (plasma) compartment (in mL), V_(C)=dose/(A+B); areaunder the curve (in μg.min.mL⁻¹). AUC=A/α+B/β; and plasma clearance (inmL.min⁻¹), clp=dose/AUC (32).

The disposition rate of stapphylokinase-related antigen from bloodfollowing bolus injection of 100 μg/kg of the selected SakSTAR variantsin groups of 4 hamsters could adequately be described by a sum of twoexponential terms by graphical curve peeling (results not shown), fromwhich the pharmacokinetic parameters t1/2α and clp, summarized in Table13 were derived. The pharmacokinetic parameters of dimericSakSTAR(K109C) and SakSTAR(K102C-PEG) were markedly different from thoseof wild type SakSTAR. Initial plasma half-lives (t1/2(α)) were 3.6 and3.0 min and plasma clearances (Clp) were 0.52 and 0.32 mL/min, fordimeric SakSTAR(K109C) and SakSTAR(K102C-PEG), respectively.

These results may be due to the increase of the Stokes radius of SakSTARas a result of the dimerization or crosslinking with PEG. According tosize-eclusion chromatography on Superdex50 by HPLC, dimericSakSTAR(K109C) and SakSTAR(K102C-PEG) have apparent molecular weights of33 kDa and 40 kDa, respectively.

Conclusion

In summary, the present experience illustrates that staphylokinasevariants with markedly reduced antibody induction but intactthrombolytic potency can be generated. To our knowledge, thisobservation constitutes the first case in which a heterologous protein,with the use of protein engineering techniques, is renderedsignificantly less immunogenic in man without reducing its biologicalactivity.

The present invention was inititated by the observation that certain“clustered charge-to-alanine” substitution variants of recombinantstaphylokinase (SakSTAR variant (9)) had a reduced reactivity withantibodies induced by treatment with wild type SakSTAR (3,4) and inducedless antibodies than wild type SakSTAR in patients with peripheralarterial occlusion (22,32,35). In an effort to optimize the specificactivity versus antigenicity radio, a comprehensive mutagenesis study,comprising the construction and expression of over 250 plasmids encodingSakSTAR variants, and the purification of the translation products wasundertaken. The SakSTAR variants were characterized in terms of specificactivity, affinity towards a panel of murine monoclonal antibodies andabsorption of SakSTAR specific antibodies from pooled plasma of 10patients treated with wild type SakSTAR and of two subpools of 3patients each which reacted strongly (subpool B) or poorly (subpool C)with the immunodominant epitope K74,E75,R77. In a later phase, anadditional pool of 40 patients treated with wild-type SakSTAR was alsoused for absorption studies. The values obtained with both pools were ingood agreement. Linear regression analysis yielded

(Pool 40)=0.84×(Pool 10)+14, with r=0.94 and n=61.

Residues for site-directed mutagenesis were selected in three ways: 1) acomprehensive analysis of variants with 1 or 2 adjacent amino acidssubstituted with Ala: 2) analysis of the differential reactivity of thetwo natural variants SakSTAR and Sak42D (which corresponds toSakSTAR(S34G,G36R,H43R) and 3) surface exposure of the residues asderived from the three dimensional structure. From these analyses,SakSTAR(K35A), SakSTAR(N95A) and SakSTAR(S103A) emerged with specificactivities ≧200 kU/mg. SakSTAR(W66A), SakSTAR(Y73A) and SakSTAR(E75A)with reduced reactivity with ≧3 of the 5 antibodies of epitope clusterI, SakSTAR(H43A) and SakSTAR(V45A) with 3 antibodies of epitope clusterII, and SakSTAR(V32A), SakSTAR(K35A), SakSTAR(D82A) and SakSTAR(K130A)and ≧3 of the 5 antibodies of epitope cluster III. However, onlySakSTAR(Y73A) and SakSTAR(K74A) reduced the antibody binding from pooledimmunized patient plasma by ≧30 percent. Analysis of the differentialreactivity of SakSTAR and Sak42D revealed that the reduced reactivitywith antibodies of epitope cluster III and with immunized patient plasmacould be ascribed to the G36R substitution. In addition E65 and K135 ofSakSTAR were targeted because of their location, in thethree-dimensional structure (36), in the close vicinity of theimmunodominant K74,E75,R77 epitope.

Substitution of K35 with other amino acids than Ala did not improve thespecific activity over that of SakSTAR(K35A), and substitution of Y73with other residues did not rescue the impaired specific activity.SakSTAR(E80A,D82A) had an intact specific activity and a somewhat lowerreactivity with immunized patient plasma, and the increased specificactivity of SakSTAR(N95A) could not be conferred to variants containingK74A or K135R. SakSTAR(K130A) was outperformed in terms of specificactivity by SakSTAR(K130T). K74 confirmed its key role in binding ofantibodies from immunized patient plasma in the absence of a markedlyreduced reactivity with the murine monoclonal antibodies (22), the bestspecific activity/antigenicity ratios being obtained with SakSTAR(K74Q)and SakSTAR(K74R).

The SakSTAR(K130T,K135R) variant was taken as a template for additivemutagenesis because of its high specific activity with a moderatereduction of binding to antibodies of epitope cluster III and ofabsorption of antibodies from immunized patient plasma (Table 6).Addition of G36R, K74R or both to the template did not affect thespecific activity, but reduced the reactivity with monoclonal antibodiesagainst epitope cluster III (G36R substitution) and decreased theabsorption of antibodies from immunized patient plasma (K74Rsubstitution). Addition of E80A and/or D82A to the SakSTAR(K130T,K135R)template did not affect the specific activity and was selected as atemplate for further mutagenesis because of its reduced reactivity withepitope cluster III antibodies (Table 7). Addition of K74R and even moreof K74Q drastically reduced the reactivity with immunized patientplasma. Finally, addition of E65D or E65Q to the SakSTAR(K74R,E80A,D82A,K130T,K135R) template yielded variants with intact specific activitywhich only bound 1/3 of the antibodies of pooled immunized patientplasma, only about 10 to 30 percent of the antibodies from plasma ofpatients with a high concentration of antibodies directed towards theimmunodominant K74,E75,R77 (Subpool B) and only about 60 percent of theantibodies from plasma of patients with a very low concentration ofantibodies directed against this immunodominant epitope (Subpool C).Based on this analysis, SakSTAR(K74Q,E80A, D82A,K130T,K135R), andSakSTAR(E65D,K74R,E80A, D82A,K130T,K135R) were selected for furtheranalysis.

The fibrinolytic potency and the fibrin-selectivity of these selectedmutants in a plasma milieu was indistinguishable from that of wild typeSakSTAR. The temperature stability of the mutants was still acceptablewith no significant loss of activity upon incubation at 37° C. for 3days, although at 56° and 70° C., they were more rapidly inactivatedthan wild-type SakSTAR. The pharmacokinetics of the SakSTAR variantsfollowing intravenous bolus injection in hamsters did not reveal majordifferences with wild type SakSTAR except for a possibly somewhat higherplasma clearance.

In conclusion, the two variants of SakSTAR which emerged from thepresent site directed mutagenesis program are characterized by an intactor slightly increased specific activity, maintained thrombolytic potencyand fibrin-selectivity in a human plasma milieu, acceptable althoughslightly reduced temperature stability and a markedly reduced reactivitywith anti-SakSTAR antibodies in pooled immunized patient plasma. In viewof the previously found correlation between reduced antigenicity andreduced immunogenicity of certain “charged-cluster-to-alanine” variantsinvestigation of immunogenicity associated with their use forthrombolytic therapy in man appeared warranted.

Highly purified, sterilized preparations ofSakSTAR(K74Q,E80A,D82A,K130T,K135R) andSakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) were produced and found tocontain low endotoxin levels, and to be devoid of acute toxicity in micefollowing intravenous bolus injection at a dose of 3 mg/kg.

Intra-arterial administration of wild-type SakSTAR.SakSTAR(K74Q,E80A,D82A,K130T, K135R) orSakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) as a bolus of 2 mg followed byan infusion of 1 mg/hr in 6 patients with angiographically documentedocclusion of a peripheral artery or bypass graft each, resulted incomplete recanalization in 10 patients and partial recanalization in 2,without measurable systemic plasminogen activation. Followingadministration of wild-type or variant SakSTAR, neutralizing antibodytiters and specific IgG levels remained low for one week. From thesecond or third week onwards, an increase of SakSTAR-neutralizingactivity to ≧5 μg/mL plasma was observed in the 3 of the 6 patientsgiven SakSTAR(K74Q,E80A,D82A,K130T,K135R), and in only 1 of the 6patients given SakSTAR(E65D,K74R,E80A,D82A,K130T,K135). Thisimmunization rate with the variants is significantly lower than theimmunization rate of 80% observed in 70 patients treated with SakSTAR(p=0.01 by 2×3 Chi square analysis). The antibodies induced by treatmentwith the SakSTAR variants were completely absorbed by SakSTAR, and bythe respective variants in all but one patient with measurableneutralizing antibody levels, indicating that immunization was not dueto neoepitopes generated by substitution but to residual epitopes in theSakSTAR template.

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TABLE 1 Alanine-to-wild-type” reversal variants of“charged-cluster-to-alanine” mutants of SakSTAR: Associations constants(K_(A) × 10⁷ mol/L⁻¹) for the binding to insolubilized murine monoclonalantibodies (Mabs), and absorption (percent) of antibodies of immunizedpatient plasma murine MAbs Exp. Spec. Act. Epitope I Epitope II EpitopeIII SakSTAR patient plasma Variant (mg/L) (kU/mg) 17G11 26A2 30A2 2B123G10 18F12 14H5 28H4 32B2 7F10 7H11 25E1 40C8 24C4 1A10 Pool Subpool BSubpool C SakSTAR 130 22 13 2.9 7.8 11 38 7.4 19 7.7 2.4 4.0 14 5.4 2.90.6 95 95 95 SakSTAR(K35A, E38A) 97 15 22 4.2 11 7.9 110 10 15 12 2.2<0.1 <0.1 <0.1 1.0 1.0 93 91 94 SakSTAR(K74A, E75A, R77A) 110 11 <.01<0.1 <0.1 <0.1 150 17 28 14 3.3 2.4 11 4.0 2.1 0.9 55 43 95SakSTAR(K35A, E38A, K74A, E75A, R77A) 50 11 <0.1 <0.1 <0.1 <0.1 110 3626 15 2.0 <0.1 <0.1 <0.1 1.5 1.2 52 41 92 SakSTAR(E38A, K74A, E75A,R77A) 43 11 <0.1 <0.2 <0.1 <0.1 140 39 26 15 2.1 <0.1 3.2 3.7 1.6 1.1 5044 95 SakSTAR(K35A, K74A, E75A, R77A) 56 9.2 <0.1 0.15 <0.1 <0.1 52 1429 8.8 2.3 <0.1 1.8 <0.1 1.8 0.8 46 43 95 SakSTAR(K35A, E38A, E75A,R77A) 44 11 0.3 0.1 0.2 <0.1 75 9.8 12 7.3 1.6 <0.1 <0.1 <0.1 0.53 0.6492 87 94 SakSTAR(K35A, E38A, K74A, R77A) 41 8.8 2.9 <0.1 2.0 0.33 110 2931 10 2.0 <0.1 <0.1 <0.1 0.63 0.74 56 50 93 SakSTAR(K35A, E38A, K74A,E75A) 19 13 <0.1 0.1 <0.1 <0.1 180 41 37 15 1.6 <0.1 <0.1 <0.1 1.2 0.4548 41 92 SakSTAR(E38A, E75A, R77A) 88 11 0.6 0.15 0.4 0.3 79 12 15 102.0 <0.1 2.6 4.7 1.1 0.81 95 88 95 SakSTAR(E38A, E75A) 66 16 0.3 <0.1<0.1 0.9 56 11 13 8.9 2.0 <0.1 20 4.8 1.3 1.6 91 90 95 SakSTAR(K35A,E75A, R77A) 68 9.2 <0.1 <0.1 <0.1 <0.1 60 7.0 13 11 3.3 <0.1 1.5 <0.10.8 1.1 88 89 95 SakSTAR(K35A, E75A) 150 17 0.12 <0.1 0.16 0.14 40 7.213 9.2 4.2 <0.1 1.8 <0.1 1.4 1.5 94 93 95 SakSTAR(K74A) 100 12 7.6 0.174.4 2.1 55 15 33 14 3.6 2.9 14 4.9 3.4 1.2 59 45 95 SakSTAR(E75A) 140 131.2 <0.1 <0.1 <0.1 46 8.5 14 12 3.4 4.5 18 5.0 1.2 2.1 95 93 95SakSTAR(K74A, E75A, R77A, E80A, D82A) 50 14 <0.1 <0.1 <0.1 <0.1 180 1933 19 3.7 <0.1 <0.1 <0.1 <0.1 1.2 49 29 89 SakSTAR(E80A, D82A) 130 7.312 2.1 6.5 5.9 79 6.1 8.4 7.8 1.9 <0.1 <0.1 <0.1 <0.1 0.44 89 83 92SakSTAR(E80A) 160 13 13 3.3 7.9 10 35 7.4 17 8.6 2.1 <0.1 16 3.6 <0.11.7 94 93 95 SakSTAR(D82A) 160 17 12 4.8 7.3 11 31 7.8 17 12 2.7 <0.10.18 <0.1 <0.1 2.3 95 93 95 SakSTAR(E75A, D82A) 170 20 15 3.1 6.6 7.2 698.1 15 14 4.9 0.17 0.7 0.5 0.1 1.4 95 95 95 Apparent associationconstants ≧ 10-fold lower than those of wild-type SakSTAR arerepresented in bold type; Spec. Act. ≧100.000 HU/mg represented in boldtype; ≦60% absorption represented in bold type.

TABLE 2 Baseline characteristics and treatment outcome of the patientswith peripheral arterial occlusion treated with SakSTAR, SakSTAR(K74A)or SakSTAR(K74A, E75A, R77A) Total dose Total Age of Length of Recanal-of throm- duration Compound Gen- Age Clinical Locus of occlusionocclusion ization by bolytic a- of infu- Additional Patient Id. der(yrs) ischemia occlusion (days) (cm) thrombolysis gent (mg) sion (hrs)therapy SakSTAR MEE F 67 Rest pain Left SFA 30 8 complete 7.0 5.0 PTAFOR M 68 Claudication Left IA (stent) 14 18 complete 6.5 4.5 PTA + stentDAN M 73 Claudication Right SFA 30 6 complete 7.5 5.5 PTA BER F 63 Restpain Left FT graft 18 55 complete 18 28 PTA DAM F 43 Acute Left brachialand 2 7 complete 19 17 PTA + stent radial anery TOR M 68 ClaudicationRight SFA (pop- 50 12 complete 6.0 4.0 PTA + liteal aneurysm)femoropopliteal bypass graft CLA M 74 Acute Left PA 1.5 20 complete 9.07.0 — MAN M 65 Acute Left EIA (stent) 4 20 complete 6.5 4.5 (amputationleft digit V) MAT M 64 Subacute Right FP graft 3 45 complete 8.0 6.0 (−)Mean ± SEM 65 ± 3.0 17 ± 5.6 21 ± 5.8 9.7 ± 1.7 9.1 ± 2.7 SakSTAR (K74A)LIE M 70 Subacute Right FF graft 10 48 complete 11 9.0 PTA ENG M 50Claudication Right SFA 28 10 complete 12 10 PTA COX F 48 ClaudicationRight PA graft 25 7 partial 15 15 PTA MAN F 68 Claudication Right SFA≧120 9 complete 9.0 7.0 PTA VHE M 47 Acute Right IF graft 10 54 complete18 16 Surgical graft revision MUL F 51 Acute Right IF and FP 1 63complete 16 20 PTA graft BUR F 67 Rest pain Right TF trunc 9.0 38partial 18 21 — NIJ F 60 Rest pain Left AF graft 23 78 complete 15 21 —POE* M 49 Subacute Right TF trunc 2 30 partial 6.0 4.0 rt-PA, surgicalgraft lengthening VBE M 39 Subacute Right BA 20 28 complete 18 23 Stentright SC (embolism) artery, first rib resection SME F 50 Subacute TFtrunc 18 32 complete 21 19 none WOL M 67 Subacute Right PA 4 25 complete16 22 — Mean ± SEM 56 ± 3.0 23 ± 9.2 35 ± 6.4 15 ± 1.2 16 ± 1.9 SakSTAR(K74A, E75A, R77A) JAC F 65 Acute Right BA and UA 0.3 5 complete 14 12 —MAE M 74 Rest pain Left SFA 10 50 complete 9.0 7.0 PTA CRA F 52Claudication Right IA and FA 14 28 complete 25 23 PTA + stent artery VDBM 68 Claudication Left SFA 90 12 complete 9.0 7.0 PTA DUN M 71 SubacuteLeft SFA 14 6 complete 9.0 7.0 PTA DEL M 59 Acute Right FT graft 3 42complete 9.0 7.0 PTA Mean ± SEM 65 ± 3.3 22 ± 14 24 ± 7.8 13 ± 2.6 11 ±2.6 AF: aortofemoral; BA: brachial artery; CIA: common iliac artery; FF:femorofibular; FP: femoropopliteal; FT: femorotibial; IA: iliac artery;IF: iliofemoral; PA: popliteal artery; PTA, percutaneous transluminalangioplasty; SFA: superficial femoral artery; TF: tibiofibular; UA:ulnar artery. *Previous treatment with SakSTAR in 1994

TABLE 3 Alanine-substitution variants of SakSTAR: Association constants(K_(A) × 10⁷ mol/L⁻¹) for binding to insolubilized murine monoclonalantibodies (Mab) and absorption (percent) of antibodies of immunizedpatient plasma murine MAbs Exp. Spec. Act. Epitope cluster I Epitopecluster II Epitope cluster III SakSTAR patient plasma Variant (mg/L)(kU/mg) 17G11 26A2 30A2 2B12 3G10 18F12 14H5 28H4 32B2 7F10 7H11 25E140C8 24C4 1A10 Pool Subpool B Subpool C SakSTAR 120 9.3 13 2.9 7.8 11 387.4 19 7.7 2.4 4.0 14 5.4 2.9 0.6 95 95 95 [6.4 17 7.4 19 35 1825 >18 >14 1.1 0.6 11 16 6.3 2.0] SakSTAR(S34G,G36R,H43R) 120 10 14 3.37.5 11 <0.1 <0.1 <0.1 20 2.7 <0.1 <0.1 <0.1 0.15 1.7 87 76 75SakSTAR(F4A) LE SakSTAR(D5A, K6A) 150 [1.3 21 12 9.2 9.7 12 23 17 10 0.41.3 3.9 0.8 4.5 3.8] 95 95 95 SakSTAR(K8A, K10A) 24 [1.8 16 5.1 29 15 2216 26 18 1.0 0.93 11 1.1 18 0.75] 95 95 95 SakSTAR(Y9A) 24 78 22 49 6.62.3 16 44 8.4 20 54 2.6 2.4 16 9.6 1.1 0.5 96 95 95 SakSTAR(K11A, D13A,D14A) LE SakSTAR(D13A) 6 46 2.4 6.1 2.0 3.7 3.4 11 1.9 4.5 4.4 8.7 1.52.4 1.1 3.6 <0.1 95 94 95 SakSTAR(D14A) 14 30 5.1 13 4.0 6.6 3.1 38 7.712 15 2.2 2.7 6.0 3.2 5.6 <0.1 95 94 95 SakSTAR(516A) 41 160 8.1 16 4.58.4 9.0 15 6.1 13 21 3.6 4.0 8.0 4.3 2.5 0.5 95 95 95 SakSTAR(Y17A,F18A) 27 30 13 22 3.3 10 9.5 21 4.6 6.7 12 2.5 1.2 18 6.5 3.4 <0.1 95 9595 SakSTAR(E19A, P20A) 36 9 11 19 3.3 9.2 12 15 6.1 11 16 1.0 1.1 15 5.13.3 <0.1 93 93 95 SakSTAR(T21A) 54 170 4.8 15 2.4 8.7 9.6 32 11 24 181.3 1.8 9.6 2.9 5.6 0.6 95 95 95 SakSTAR(P23A) 41 67 14 31 4.4 14 22 415.3 37 13 10 4.0 11 7.6 3.1 1.9 91 95 95 SakSTAR(Y24A) 10 40 17 33 4.313 11 33 4.3 7.0 12 4.0 0.4 14 6.8 4.8 <0.1 95 95 95 SakSTAR(L25A) LESakSTAR(M26A) LE SakSTAR(V27A) 62 50 3.3 15 1.6 7.8 7.4 12 2.9 3.7 332.0 1.3 5.9 2.8 4.2 1.2 95 95 95 SakSTAR(N28A) 90 <5 5.8 29 2.1 7.0 5.527 10 20 2.5 2.1 2.1 5.6 2.1 2.1 0.7 95 95 95 SakSTAR(N28A, V29A) 32 4518 30 2.5 20 18 20 14 20 24 2.7 3.3 20 1.1 5.0 2.0 93 95 95SakSTAR(T30A) 52 140 7.4 13 2.1 7.0 6.1 7.6 3.4 5.1 13 3.3 5.6 12 3.45.4 0.8 94 95 95 SakSTAR(V32A) 78 45 10 9.6 2.4 6.2 7.8 56 17 12 14 1.4<0.1 <0.1 <0.1 <0.1 2.2 90 93 95 SakSTAR(D33A, K35A) 130 [2.1 19 14 1419 15 24 32 10 5.3 1.4 5.1 3.8 5.1 3.0] 95 95 95 SakSTAR(S34A) 29 110 1724 4.6 9.5 11 28 11 22 15 2.9 3.1 8.8 3.8 2.0 0.2 95 95 93 SakSTAR(K35A)230 6.4 14 3.3 8.0 7.4 31 11 12 11 2.6 <0.1 1.3 0.2 1.7 0.8 91 88 95SakSTAR(K35A, E38A) 97 15 22 4.2 11 7.9 110 10 15 12 2.2 <0.1 <0.1 <0.11.0 1.0 93 91 94 SakSTAR(G36A) 14 72 3.5 9.8 1.5 5.7 6.5 52 4.2 17 9.21.4 <0.1 <0.1 0.9 5.0 1.0 86 83 78 SakSTAR(N37A) 40 110 5.6 31 3.0 10 1120 4.1 14 10 2.9 1.4 5.3 3.5 3.6 0.8 95 95 95 SakSTAR(L39A, L40A) 8 <514 15 3.1 5.1 8.0 27 16 6.3 12 2.7 1.2 5.4 3.2 2.1 0.9 93 93 95SakSTAR(S41A, P42A) 24 48 10 25 4.1 13 12 11 3.0 1.9 27 2.7 3.2 15 4.83.6 1.1 95 95 95 SakSTAR(H43A) 37 69 15 28 9.7 18 7.6 <0.1 <0.1 <0.1 9.11.5 2.0 23 7.8 7.2 1.6 95 95 95 SakSTAR(H43A, Y44A) 13 <5 10 22 3.7 1715 <0.1 <0.1 <0.1 1.4 3.0 2.3 11 5.2 2.1 0.1 95 95 95 SakSTAR(V45A) 19<5 16 5.6 1.4 4.8 6.3 2.2 0.2 1.7 32 2.6 2.1 8.3 1.1 2.8 1.6 91 92 95SakSTAR(E46A, K50A) LE SakSTAR(F47A) 6 <5 <0.1 4.0 1.0 3.9 3.4 5.7 2.72.8 8.5 1.7 0.9 8.8 3.0 3.0 0.9 90 82 97 SakSTAR(I49A) 2 43 2.7 27 7.823 22 35 4.4 11 6.2 1.1 2.0 5.7 2.0 1.7 0.6 95 95 95 SakSTAR(K50A) 15 42<0.1 13 2.9 7.8 8.7 46 8.3 12 2.2 0.5 2.8 6.4 4.0 2.3 0.6 95 94 95SakSTAR(T53A, T54A) 14 68 0.9 19 2.7 7.6 7.8 41 6.7 12 15 1.5 1.9 5.12.3 1.0 0.6 93 94 95 SakSTAR(L55A) LE SakSTAR(T56A) 17 150 5.5 15 3.2 1213 56 5.3 12 11 2.0 3.5 6.1 2.7 4.3 0.2 94 92 95 SakSTAR(K57A, E58A,K59A) 94 [4 8.7 6.0 7.3 27 16 14 6.7 5.6 0.52 0.36 1.7 0.42 1.0 1.1]SakSTAR(160A) 11 96 12 20 2.9 11 13 27 6.4 25 2.7 1.5 0.7 5.8 2.9 1.71.0 95 95 95 SakSTAR(E61A, E65A) 80 [9.5 >10 8.8 21 29 >11 >16 6.6 >7.24.6 0.5 4.6 2.0 5.9 1.5] SakSTAR(Y62A, Y63A) 24 <5 <0.1 4.3 0.3 2.1 1.911 2.2 3.1 8.4 1.7 0.6 9.2 3.6 3.8 0.7 89 83 95 SakSTAR(Y63A) 4 <5 <0.118 3.7 9.6 13 17 5.3 3.3 15 1.1 2.2 5.3 4.3 1.0 3.7 89 82 95SakSTAR(V64A) 14 48 14 16 2.9 6.3 7.8 15 15 21 21 2.6 1.6 7.6 5.6 2.80.7 94 92 95 SakSTAR(E65A) 25 97 53 20 4.4 12 7.0 10 5.6 9.7 1.9 1.8 2.34.7 3.0 5.8 0.97 95 95 95 SakSTAR(E65A, D69A) <5 SakSTAR(W66A) 26 <5<0.1 <0.1 <0.1 <0.1 <0.1 15 4.4 5.7 23 3.3 2.0 10 1.4 1.8 0.8 85 78 92SakSTAR(L68A) 16 93 14 22 3.5 8.5 9.3 29 8.7 16 15 4.0 2.1 5.3 3.6 4.1<0.1 92 92 95 SakSTAR(T71A) LE SakSTAR(Y73A) 20 <5 9.5 <0.1 <0.1 <0.14.8 33 7.3 11 8.9 2.0 0.5 9.0 2.5 1.4 0.8 63 44 93 SakSTAR(Y73A, K74A)24 <5 18 <0.1 <0.1 <0.1 <0.1 19 6.7 23 9.9 3.2 2.7 13 4.0 1.6 1.1 47 2887 SakSTAR(K74A) 80 69 4.4 2.7 0.2 2.2 1.1 17 5.2 14 7.6 2.2 2.0 6.8 3.31.8 0.9 64 58 95 SakSTAR(K74A, E75A, R77A) 68 9.2 <0.1 <0.1 <0.1 <0.1 607.0 13 11 3.3 <0.1 1.5 <0.1 0.8 1.1 88 89 95 SakSTAR(K74A, R77A) 34 413.5 1.8 0.2 1.5 0.4 23 2.4 10 2.1 1.8 1.7 2.3 2.2 1.2 0.7 74 60 95SakSTAR(E75A) 140 13 1.2 <0.1 <0.1 <0.1 46 8.5 14 12 3.4 4.5 18 5.0 1.22.1 95 93 95 SakSTAR(F76A) 9 90 20 9.6 1.0 2.7 3.9 13 6.2 20 15 1.7 0.35.9 2.1 1.2 1.0 94 92 95 SakSTAR(V78A, V79A) 23 68 12 23 4.0 10 17 21 1834 28 2.3 1.6 4.7 <0.1 0.5 1.7 93 93 95 SakSTAR(E80A) 160 13 13 3.3 7.910 35 7.4 17 8.6 2.1 <0.1 16 3.6 <0.1 1.7 94 93 95 SakSTAR(E80A, D82A)130 7.3 12 2.1 6.5 5.9 79 6.1 8.4 7.8 1.9 <0.1 <0.1 <0.1 <0.1 0.4 89 8392 SakSTAR(L81A) 23 28 12 33 1.6 40 11 52 11 17 17 3.9 1.4 5.2 7.1 4.61.5 88 95 95 SakSTAR(D82A) 160 17 12 4.8 7.3 11 31 7.8 17 12 2.7 <0.10.2 <0.1 <0.1 2.3 95 93 95 SakSTAR(D82A, S84A) 72 130 8.3 14 2.6 8.1 8.523 3.8 12 11 1.7 <0.1 <0.1 1.4 <0.1 1.0 91 91 95 SakSTAR(S84A) 12/26 898.0 16 3.8 8.6 10 90 8.3 11 36 1.8 2.2 1.6 3.0 3.5 0.5 95 95 95SakSTAR(K86A, F88A) 73 [7.2 1.4 3.7 6.0 4.6 5.7 4.9 7.7 15 4.4 <0.1 5.40.80 1.9 0.13 SakSTAR(I87A) 18 98 6.7 23 2.8 8.6 9.1 10 3.6 11 7.4 2.71.1 7.8 3.4 4.5 1.0 95 95 95 SakSTAR(V89A) 20 87 4.6 11 2.6 6.6 2.2 287.2 7.3 3.0 1.3 1.2 5.1 2.9 3.1 0.83 95 95 95 SakSTAR(T90A) 78 120 6.012 0.9 3.7 3.1 20 4.8 7.2 <0.1 <0.1 2.1 6.6 2.6 2.1 0.5 95 95 95SakSTAR(Y91A) 5 53 6.0 16 3.0 7.0 13 28 8.2 16 0.6 2.1 1.4 3.7 1.6 1.60.2 95 95 95 SakSTAR(Y92A) 16 120 16 23 4.1 13 12 29 7.3 18 <0.1 1.7 4.410 3.9 5.9 1.1 95 95 95 SakSTAR(E93A, K94A) 97 [8.2 19 13 30 24 1811 >10 9.0 0.88 1.4 11 2.4 7.0 2.1] SakSTAR(K94A, N95A, K97A) 32 8 NT 9594 95 SakSTAR(N95A) 25 260 10 18 4.0 10 11 50 13 14 4.9 2.3 3.7 7.3 4.72.9 0.8 95 94 95 SakSTAR(K96A, K97A, K98A) 47 [2.8 41 23 37 90 >16 9.119 16 0.41 0.58 17 1.2 13 0.30] SakSTAR(E99A) 24 42 7.4 15 4.0 8.4 8.922 2.7 4.7 <0.1 <0.1 2.1 6.2 7.3 14 0.8 92 91 92 SakSTAR(E99A, E100A) LESakSTAR(T101A) 23 85 4.6 11 2.1 6.6 7.3 30 2.8 16 0.7 1.0 1.3 5.4 2.42.9 0.8 95 95 95 SakSTAR(K102A) 12 89 4.9 12 3.7 6.5 6.3 30 3.1 15 8.26.1 0.8 4.2 1.9 10 0.6 95 93 95 SakSTAR(S103A) 67 210 9.0 16 5.0 9.4 9.119 5.9 13 13 3.6 3.9 8.3 4.7 2.8 0.9 94 95 95 SakSTAR(F104A) 14 55 5.819 4.8 14 27 7.3 5.0 14 4.8 <0.1 0.4 7.6 3.4 1.1 1.3 95 93 95SakSTAR(I106A) 2 93 2.3 13 3.0 7.4 6.7 5.5 5.2 17 11 1.4 1.8 3.1 1.8 1.20.5 95 95 95 SakSTAR(T107A) 32 130 5.2 15 3.4 9.8 10 32 8.7 4.7 14 1.93.1 6.3 3.2 5.0 0.8 94 94 95 SakSTAR(E108A, K109A) 170 [1.6 5.1 7.2 195.1 28 15 21 21 1.2 0.43 6.9 1.4 10 1.9 SakSTAR(F111A) 5 49 3.7 16 3.813 22 21 8.4 12 3.1 0.8 2.8 2.9 1.5 1.5 0.9 95 95 95 SakSTAR(V112A,V113A) 64 130 4.2 16 3.9 10 12 34 5.8 13 8.0 0.3 1.5 4.3 2.3 3.0 0.8 9595 95 SakSTAR(D115A, S117A) 80 54 3.3 14 4.1 15 15 17 3.4 19 0.7 <0.11.5 4.8 2.6 1.3 0.9 95 95 95 SakSTAR(D115A, E118A, H119A) 32 [2.5 32 3.421 8.7 13 9.9 23 9.3 1.2 1.0 24 2.1 9.0 1.8 SakSTAR(L116A, S117A) 25 <54.4 35 3.6 33 42 160 29 220 <0.1 <0.1 0.5 4.1 4.9 3.5 1.6 94 95 95SakSTAR(H119A, K121A) 130 [8.0 24 11 26 29 25 14 29 12 0.52 1.2 11 2.920 1.2 SakSTAR(I120A) 26 75 23 26 5.1 17 16 30 9.8 25 9.0 6.9 3.0 15 5.15.2 1.0 93 95 95 SakSTAR(N122A) 5 19 NT 95 93 95 SakSTAR(F125A) 3 <102.8 18 4.7 11 18 17 3.2 6.0 1.9 <0.1 0.3 5.3 2.1 0.9 1.6 93 90 95SakSTAR(N126V) 11 51 7.6 13 2.0 12 13 30 58 290 9.8 2.5 1.8 8.0 .2 6.50.7 95 95 95 SakSTAR(L127A) 11 54 8.9 6.7 1.8 5.0 6.6 25 4.9 14 8.4 1.50.9 1.9 0.9 2.5 1.8 93 94 95 SakSTAR(I128A) 10 20 16 25 4.8 15 14 38 2.64.3 8.2 2.9 2.5 2.0 4.2 6.7 0.9 95 93 95 SakSTAR(T129A) 44 190 5.3 152.3 14 24 21 13 15 4.2 2.3 0.7 10 3.3 1.3 1.0 95 95 95 SakSTAR(K130A)130 280 5.1 12 3.2 6.4 3.5 22 6.7 11 15 2.7 <0.1 <0.1 4.1 0.9 0.6 92 7471 SakSTAR(V131A) 130 70 6.5 17 2.9 11 13 19 14 19 29 3.4 1.9 13 5.3 8.60.9 95 95 95 SakSTAR(V132A) 100 130 4.2 15 2.6 9.2 11 33 12 30 19 2.12.1 3.6 <0.1 2.6 0.4 95 95 95 SakSTAR(I133A) 3 99 9.4 15 1.9 7.8 7.8 246.0 9.1 8.6 1.4 0.56 6.4 1.6 1.6 0.9 95 95 95 SakSTAR(E134A, K135A,K136A) 74 [22 21 6.7 25 25 >18 >25 >15 >12 1.7 0.2 11 0.94 6.0 2.6]SakSTAR(K135A) 54 410 5.2 12 11 7.9 11 20 11 11 3.8 2.0 1.6 6.9 3.7 1.90.9 95 95 95 SakSTAR(K136A) 180 7.6 18 5.6 12 13 54 5.3 12 16 3.2 3.58.6 3.9 1.8 0.5 91 83 95 LE: expression level below 3 mg/L

TABLE 4 Mutagenesis of S34, G36 and H43: Association constants (K_(A) ×10⁷ mol/L⁻¹) for binding to insolubilized murine monoclonal antibodies(Mab) and absorption (percent) of antibodies of immunized patient plasmamurine MAbs Exp. Spec. Act. Epitope cluster I Epitope cluster II Epitopecluster III SakSTAR patient plasma Variant (mg/L) (kU/mg) 17G11 26A230A2 2B12 3G10 18F12 14H5 28H4 32B2 7F10 7H11 25E1 40C8 24C4 1A10 PoolSubpool B Subpool C SakSTAR 120 9.3 13 2.9 7.8 11 38 7.4 19 7.7 2.4 4.014 5.4 2.9 0.6 95 95 95 SakSTAR(S34G, G36R, H43R) 120 10 14 3.3 7.5 11<0.1 <0.1 <0.1 20 2.7 <0.1 <0.1 <0.1 0.15 1.7 87 76 75 SakSTAR(S34A) 29110 17 24 4.6 9.5 11 28 11 22 15 2.9 3.1 8.8 3.8 2.0 0.2 95 95 93SakSTAR(G36A) 14 72 3.5 9.8 1.5 5.7 6.5 52 4.2 17 9.2 1.4 <0.1 <0.1 0.95.0 1.0 86 83 78 SakSTAR(G36E) 12 66 3.1 8.7 1.4 4.8 4.7 12 2.8 6.1 7.61.0 <0.1 <0.1 <0.1 3.4 1.1 89 83 72 SakSTAR(G36K) 34 88 9.9 23 3.1 8.39.8 21 3.9 13 15 3.0 <0.1 <0.1 <0.1 2.6 1.2 88 80 69 SakSTAR(G36L) 15 923.6 11 1.8 6.1 6.1 16 1.4 6.4 12 1.3 <0.1 <0.1 <0.1 0.6 1.1 93 85 72SakSTAR(G36N) 10 91 8.7 10 1.6 6.1 6.2 33 3.3 7.8 7.9 1.8 <0.1 <0.1 1.00.3 0.5 86 80 75 SakSTAR(G36Q) 21 92 10 12 1.8 6.7 6.5 23 3.8 7.5 7.31.5 <0.1 <0.1 <0.1 0.1 0.4 87 84 73 SakSTAR(G36R) 45 100 11 2.4 3.3 1010 27 4.6 14 20 3.4 <0.1 <0.1 <0.1 3.1 1.2 89 81 70 SakSTAR(H43A) 37 6915 28 9.7 18 7.6 <0.1 <0.1 <0.1 9.1 1.5 2.0 23 7.8 7.2 1.6 95 95 95SakSTAR(H43R) 43 120 15 11 2.7 7.6 11 <0.1 <0.1 <0.1 13 6.4 0.7 18 6.75.7 1.4 95 95 95 SakSTAR(S34G, G36R) 45 90 3.1 12 2.3 4.8 4.2 13 8.3 249.1 1.9 <0.1 <0.1 <0.1 <0.1 0.6 92 83 69 SakSTAR(S34G, G36R, H43R, K74A)1 12 12 4.7 3.8 7.4 9.4 <0.1 0.1 0.6 25 2.3 <0.1 <0.1 0.5 0.8 1.7 67 5683 SakSTAR(S34G, G36R, K74A) 15 26 4.0 2.1 <0.1 1.8 0.6 10 2.2 15 13 1.8<0.1 <0.1 <0.1 0.3 2.2 59 28 68 SakSTAR(K35G, G36R, H43D) 32 6 1.8 1.51.6 2.0 8.9 <0.1 <0.1 <0.1 7.1 1.3 <0.1 <0.1 <0.1 <0.1 0.9 82 75 72SakSTAR(G36R, K74A) 40 35 19 7.0 0.2 4.3 2.0 53 27 28 19 4.4 <0.1 <0.1<0.1 1.2 1.0 48 33 58 SakSTAR(G36R, K74R) 68 150 4.7 17 3.8 11 8.0 166.0 6.4 3.0 1.6 <0.1 <0.1 <0.1 0.2 0.8 81 54 73 SakSTAR(G36R, K74A,N95A) 11 25 6.1 2.9 2.9 2.4 <0.1 31 5.7 12 5.3 4.1 <0.1 <0.1 <0.1 <0.10.9 53 32 63 SakSTAR(G36R, K74A, K135R) 20 33 5.8 3.4 <0.1 1.7 0.7 26 1614 17 1.2 <0.1 <0.1 <0.1 0.4 0.5 64 32 68 SakSTAR(G36R, K74R, K135R) 4875 6.1 17 5.8 10 3.3 31 14 13 5.7 2.3 <0.1 <0.1 <0.1 0.3 0.8 77 49 68

TABLE 5 Mutagenesis of K35, Y73, K74, E80/D82, K130, V132 and K135:Association constants (K_(A) × 10⁷ mol/L⁻¹) for binding to insolubilizedmurine monoclonal antibodies (Mab) and absorption (percent) ofantibodies of immunized patient plasma murine MAbs Exp. Spec. Act.Epitope cluster I Epitope cluster II Epitope cluster III SakSTAR patientplasma Variant (mg/L) (kU/mg) 17G11 26A2 30A2 2B12 3G10 18F12 14H5 28H432B2 7F10 7H11 25E1 40C8 24C4 1A10 Pool Subpool B Subpool C SakSTAR 1209.3 13 2.9 7.8 11 38 7.4 19 7.7 2.4 4.0 14 5.4 2.9 0.6 95 95 95SakSTAR(S34G, G36R, H43R) 120 10 14 3.3 7.5 11 <0.1 <0.1 <0.1 20 2.7<0.1 <0.1 <0.1 0.15 1.7 87 76 75 SakSTAR(K35A) 230 6.4 14 3.3 8.0 7.4 3111 12 11 2.6 <0.1 1.3 0.2 1.7 0.8 91 88 95 SakSTAR(K35E) 75 160 4.8 100.6 2.7 2.7 21 5.7 9.1 4.8 1.0 <0.1 <0.1 <0.1 1.5 0.4 95 95 92SakSTAR(K35Q) 9 69 3.2 9.5 1.3 5.2 5.4 22 2.7 9.3 8.5 1.2 0.5 1.7 <0.11.9 1.0 95 95 95 SakSTAR(Y73A) 20 <5 9.5 <0.1 <0.1 <0.1 4.8 33 7.3 118.9 2.0 0.5 9.0 2.5 1.4 0.8 63 44 93 SakSTAR(Y73F) 6 31 7.9 12 1.3 2.18.0 24 15 19 15 2.6 1.6 6.2 3.5 7.2 1.4 93 95 95 SakSTAR(Y73H) 30 <5 7.31.5 <0.1 <0.1 1.4 20 7.5 17 42 4.3 3.5 6.8 5.9 9.7 1.3 76 65 95SakSTAR(Y73L) 33 <5 11 <0.1 <0.1 <0.1 <0.1 84 19 26 53 3.4 4.2 7.1 4.23.4 1.0 81 60 94 SakSTAR(Y735) 31 <5 7.0 3.6 0.59 0.6 3.0 21 8.3 14 213.2 2.2 6.7 1.3 1.3 0.7 86 69 95 SakSTAR(Y73W) 6 27 4.8 9.0 4.6 4.6 118.4 4.5 11 8.0 2.7 2.9 5.0 3.0 3.8 1.3 73 53 93 SakSTAR(K74A) 80 69 4.42.7 0.2 2.2 1.1 17 5.2 14 7.6 2.2 2.0 6.8 3.3 1.8 0.9 64 58 95SakSTAR(K74E) 12 <5 2.2 0.6 <0.1 0.7 0.1 14 1.8 7.5 9.3 1.2 2.0 3.0 1.20.6 1.0 83 43 90 SakSTAR(K74N) 9 39 2.9 4.7 1.1 3.3 1.7 10 1.6 4.8 131.5 1.9 4.0 1.8 1.4 0.9 63 46 95 SakSTAR(K74Q) 64 110 5.3 5.8 <0.1 2.51.1 24 5.9 12 5.4 1.1 2.0 6.2 2.3 2.0 0.4 70 62 94 SakSTAR(K74R) 44 1502.1 7.5 2.0 4.1 4.2 24 6.9 8.0 8.3 1.3 2.2 7.8 3.2 2.1 0.5 75 70 95SakSTAR(E80A, D82A) 130 7.3 12 2.1 6.5 5.9 79 6.1 8.4 7.8 1.9 <0.1 <0.1<0.1 <0.1 0.4 89 83 92 SakSTAR(E80A) 160 13 13 3.3 7.9 10 35 7.4 17 8.62.1 <0.1 16 3.6 <0.1 1.7 94 93 95 SakSTAR(D82A) 160 17 12 4.8 7.3 11 317.8 17 12 2.7 <0.1 0.2 <0.1 <0.1 2.3 95 93 95 SakSTAR(N95A) 25 260 10 184.0 10 11 50 13 14 4.9 2.3 3.7 7.3 4.7 2.9 0.8 95 94 95 SakSTAR(N95E) 1279 2.8 8.5 1.3 5.2 5.4 17 2.7 10 5.2 1.1 0.5 4.0 1.7 1.8 0.6 95 92 95SakSTAR(N95G) 20 160 4.1 11 1.5 6.8 7.6 36 3.3 15 3.6 1.5 0.7 5.8 2.72.7 0.9 95 90 95 SakSTAR(N95K) 54 180 9.5 14 3.2 9.0 11 51 5.0 18 4.82.5 1.6 8.3 2.9 4.8 1.1 95 95 95 SakSTAR(N95R) LE SakSTAR(K130A) 280 5.112 3.2 6.4 3.5 22 6.7 11 15 2.7 <0.1 <0.1 4.1 0.9 0.6 92 74 71SakSTAR(K130T) 290 7.8 14 3.1 8.0 5.6 43 5.4 9.7 17 2.7 <0.1 <0.1 3.40.5 0.5 92 84 82 SakSTAR(V132A) 102 130 4.2 15 2.6 9.2 11 33 12 30 192.1 2.1 3.6 <0.1 2.6 0.4 95 95 95 SakSTAR(V132L) 126 120 4.8 14 2.3 8.09.7 67 10 48 20 2.5 2.0 11 <0.1 4.8 0.4 95 95 95 SakSTAR(V132T) 78 1404.5 13 2.4 7.8 9.0 39 10 25 16 2.0 1.1 2.0 <0.1 <0.1 0.4 95 95 95SakSTAR(V132N) 16 150 4.5 11 1.7 7.0 7.2 21 17 20 15 2.0 1.5 1.3 <0.14.2 0.7 95 95 95 SakSTAR(V132R) 76 230 5.4 12 0.8 3.3 3.2 23 5.5 7.8 4.81.1 2.1 3.4 <0.1 0.4 0.4 95 95 95 SakSTAR(K135A) 54 410 5.2 12 11 7.91.1 20 11 11 3.8 2.0 1.6 6.9 3.7 1.9 0.9 95 95 95 SakSTAR(K135F) 68 643.9 6.3 1.0 6.1 4.1 13 4.5 8.4 16 2.1 1.6 5.8 1.9 1.5 0.9 95 95 95SakSTAR(K135R) 54 230 4.0 14 1.4 9.3 5.0 31 8.1 13 23 1.9 2.4 2.4 2.62.1 0.5 95 95 95 SakSTAR(K35A, K74A) 20 130 NT 84 50 95 SakSTAR(Y73A,K74A) 24 <5 18 <0.1 <0.1 <0.1 <0.1 19 6.7 23 9.9 3.2 2.7 13 4.0 1.6 1.147 28 87 SakSTAR(Y73F, K74A) 21 6 17 6.7 <0.1 3.1 <0.1 42 19 21 20 4.91.9 2.8 6.6 2.1 0.8 51 34 90 SakSTAR(I60A, K74A, N95A) 84 5.3 2.7 <0.12.5 5.3 17 7.2 9.6 3.4 2.3 2.2 6.2 2.7 2.9 0.8 60 47 95 SakSTAR(N95A,K135R) 120 240 4.9 13 1.9 9.0 9.9 18 12 15 2.5 1.5 1.8 5.9 3.6 3.7 0.895 95 95 SakSTAR(K130T, K135R) 15 280 3.7 10 1.6 7.2 8.0 13 7.7 4.5 3.71.6 <0.1 <0.1 2.4 0.4 0.6 89 60 73

TABLE 6 Combination mutants of SakSTAR(K130T, K135R) with K35A, G36R,E65X, K74X and selected other amino acids Spec. Exp Act. murine MAbsSakSTAR patient plasma (mg/ (kU/ Epitope cluster I Epitope cluster IIEpitope cluster III Pool Subpool Subpool Pool Variant mL) mg) 17G11 26A230A2 2B12 3G10 18F12 14H5 28H4 32B2 7F10 7H11 25E1 40C8 24C4 1A10 10 B C40 Code SakSTAR(K130T, K135R) 15 280 3.7 10 1.6 7.2 8.0 13 7.7 4.5 3.71.6 <0.1 <0.1 2.4 0.4 0.6 89 60 73 90  SY2 SakSTAR(G36R, K130T, K135R)26 220 7.2 18 2.8 11 14 17 3.9 8.9 5.1 1.6 <0.1 <0.1 <0.1 <0.1 1.1 79 6569 —  SY3 SakSTAR(K74R, K130T, K135R) 18 310 7.3 27 5.9 16 18 17 3.7 115.1 1.5 <0.1 <0.1 3.2 0.7 0.9 76 49 69 78  SY4 SakSTAR(K74Q, K130T,K135R) 64 190 4.0 7.2 3.0 7.7 0.9 20 5.7 7.2 8.4 1.8 <0.1 <0.1 2.7 0.71.0 50 25 67 62 SY41 SakSTAR(G36R, K74R, K130T, K135R) 5 210 7.6 26 5.717 19 17 4.7 10 5.0 1.4 <0.1 <0.1 <0.1 <0.1 0.8 73 44 69 75  SY5SakSTAR(G36R, K74Q, K130T, K135R) 88 120 5.5 7.3 0.8 11 6.4 35 11 7.16.1 2.6 <0.1 <0.1 <0.1 <0.1 0.8 51 25 63 54 SY42 SakSTAR(G36R, H43R,K74R, K130T, K135R) 29 160 5.6 8.8 2.2 10 10 <0.1 <0.1 <0.1 6.3 2.0 <0.1<0.1 <0.1 <0.1 0.9 72 39 69 —  SY9 SakSTAR(S34G, G36R, K74Q, K130T,K135R) 40 76 4.8 5.9 0.5 13 1.5 18 8.7 7.5 5.1 2.1 <0.1 <0.1 <0.1 <0.11.1 52 25 65 61 SY43 SakSTAR(E65A, K74Q, K130T, K135R) 46 170 11 9.0 2.311 13 19 66 12 5.7 2.3 <0.1 <0.1 3.5 1.2 0.6 45 16 77 55 SY45SakSTAR(G36R, E65A, K74Q, K130T, K135R) 80 83 4.7 12 1.2 16 21 27 12 106.9 2.6 <0.1 <0.1 <0.1 <0.1 0.7 44 18 65 46 SY44 SakSTAR(G36R, E65A,K74A, K130A, K135R) 17 71 5.7 10 1.8 13 12 21 8.4 6.9 4.6 1.8 <0.1 <0.1<0.1 <0.1 1.0 41 14 64 50 SY59 SakSTAR(E65A, A72S, K74Q, K130T, K135R)60 96 5.6 6.0 2.0 3.6 4.9 21 8.6 8.8 2.8 2.5 <0.1 <0.1 3.5 1.7 1.1 51 1366 56 SY51 SakSTAR(E65Q, K74Q, K130T, K135R) 40 150 6.7 18 2.1 15 (6 9.03.1 4.1 6.3 2.3 <0.1 <0.1 3.8 0.9 0.6 53 29 67 65 SY49 SakSTAR(K74Q,K86A, K130T, K135R) 54 130 2.4 4.9 <0.1 7.4 3.8 19 8.7 7.6 4.7 1.9 <0.1<0.1 3.5 1.3 1.5 56 32 69 61 SY55 SakSTAR(E65Q, T71S, K74Q, K130T,K135R) 32 210 6.2 13 1.8 10 13 11 3.9 5.0 68 23 <0.1 <0.1 3.1 1.1 0.8 4921 64 59 SY65 SakSTAR(E65Q, K74Q, E75A, K130T, K135R) 36 46 7.7 <0.1<0.1 <0.1 <0.1 10 3.9 3.2 7.4 2.6 <0.1 <0.1 4.6 1.3 0.6 43 15 62 55 SY66SakSTAR(E65Q, K74Q, E75D, K130T, K135R) 35 67 7.0 <0.1 <0.1 <0.1 <0.1 135.4 4.9 6.6 2.5 <0.1 <0.1 3.2 1.3 1.0 49 29 63 57 SY67 SakSTAR(K74Q,K130T, K135R, K136A, +137A) 19 78 4.3 24 <0.1 2.7 5.6 20 9.6 7.5 5.6 2.2<0.1 <0.1 1.7 1.1 1.2 37 12 57 50 SY68 SakSTAR(K74Q, K130A, K135R) 28240 5.6 5.4 0.5 7.5 5.3 21 8.4 14 6.1 2.4 <0.1 <0.1 4.1 2.6 0.7 57 27 7865 SY56 SakSTAR(E65Q, K74Q, K130A, K135R) 60 230 6.0 14 2.4 15 17 9.03.3 5.8 6.3 2.3 <0.1 <0.1 4.3 2.0 0.6 51 32 73 58 SY69 SakSTAR(K74Q,K130E, K135R) 46 300 4.1 4.4 0.8 6.6 3.8 18 8.5 7.8 5.2 2.1 <0.1 <0.12.4 0.7 0.9 55 29 64 59 SY57 SakSTAR(E65Q, K74Q, K130A, K135A) 88 1705.3 8.9 1.7 7.7 11 16 3.4 5.8 6.2 2.4 <0.1 <0.1 3.5 2.4 0.7 55 27 79 55SY70 SakSTAR(K74Q, K130E, V132R, K135R) 68 170 4.5 4.9 0.4 6.0 5.2 9.04.9 4.3 5.6 2.4 <0.1 <0.1 <0.1 <0.1 0.6 51 20 63 56 SY58 SakSTAR(E65Q,K74Q, T90A, K130A, K135R) 36 170 6.2 13 1.8 12 14 7.3 2.5 3.8 <0.1 <0.1<0.1 <0.1 4.1 1.9 0.5 51 27 69 57 SY71 SakSTAR(E65Q, K74Q, N95A, K130A,K135R) 40 220 6.1 14 1.9 17 15 9.0 3.3 5.8 6.3 2.3 <0.1 <0.1 4.1 2.2 0.552 29 74 58 SY72 SakSTAR(E65Q, K74Q, E118A, K130A, K135R) 86 180 8.5 182.8 15 27 11 4.1 5.7 7.3 2.6 <0.1 <0.1 6.1 2.8 0.5 50 28 72 58 SY73SakSTAR(E65Q, K74Q, N95A, E118A, K130A, 33 190 7.8 18 2.4 7.7 21 20 3.96.1 6.6 2.3 <0.1 <0.1 5.8 2.5 0.5 48 27 74 58 SY74 K135R) SakSTAR(N95A,K130A, K135R) 85 410 6.1 11 3.3 18 15 37 5.9 9.6 6.8 2.5 <0.1 <0.1 4.53.0 0.6 93 81 82 94 INT1 SakSTAR(K35A, E65Q, K74Q, K130A, K135R) 29 110NT 49 26 61 45 SY75 SakSTAR(K35A, H43R, E65Q, K74Q, K130A, 14 NT 49 2373 55 SY76 K135R) SakSTAR(E65Q, K74Q, S103A, K130A, K135R) 32 60 6.7 152.6 14 16 8.0 2.7 3.9 6.3 2.3 <0.1 <0.1 4.6 1.6 0.6 55 27 75 61 SY77SakSTAR(T21A, K35A, E65Q, K74Q, K130A, 110 NT 50 26 72 50 SY78 K135R)SakSTAR(T56A, E65Q, K74Q, K130T, K135R) 180 NT 52 31 61 55 SY79SakSTAR(K57A, E58A, E61A, K74Q, K130T, 120 NT 57 24 61 54 SY80 K135R)SakSTAR(E65Q, K74Q, K109A, K130T, K135R) 40 210 7.3 15 2.1 12 12 14 2.54.0 5.8 2.3 <0.1 <0.1 3.4 1.8 0.7 50 22 68 51 SY81 SakSTAR(E65Q, K74Q,E108A, K130T, K135R) 120 51 24 61 54 SY82 SakSTAR(E65Q, K74Q, E108A,K109A, K130T, 62 180 9.3 13 1.4 13 17 17 3.0 4.1 6.8 2.5 <0.1 <0.1 3.72.6 0.5 55 21 67 50 SY83 K135R) SakSTAR(E65Q, K74Q, K121A, K130T, K135R)73 150 5.7 13 1.5 11 14 22 3.1 4.6 1.2 <0.1 <0.1 <0.1 3.5 1.8 0.9 61 2569 57 SY85 SakSTAR(E19A, E65Q, K74Q, K130T, K135R) 3 NT 51 27 62 56 SY86SakSTAR(E65Q, K74Q, D115A, K130T, K135R) 57 NT 52 25 62 SY87SakSTAR(G36R, E65A, K74Q, K130E, V132R, 48 60 7.6 9.9 1.4 11 14 42 19 174.3 1.0 <0.1 <0.1 <0.1 <0.1 0.9 44 17 70 44 SY60 K135R) SakSTAR(E65Q,K74Q, N95A, E118A, K130A,   120 45 30 74 60 SY93 K135R, +137A)SakSTAR(E65Q, K74Q, N95A, E118A, K130A, 1,400 37 16 70 54 SY94 K135R,K136A, +137K) Association constants ≧ 10-fold lower and antibodyabsorption ≦60 percent of wild-type SakSTAR are represented in boldtype; >100,000 HU/mg represented in bold type. NT: not tested.

TABLE 7 Combination mutants of SakSTAR(E80A, D82A, K130T, K135R) withK35A, G36R, E65X, K74X, and selected other amino acids Exp murine MAbsSakSTAR patient plasma (mg/ Spec. Act. Epitope cluster I Epitope clusterII Epitope cluster III Pool Subpool Subpool Pool Variant mL) (kU/mg)17G11 26A2 30A2 2B12 3G10 18F12 14H5 28H4 32B2 7F10 7H11 25E1 40C8 24C41A10 10 B C 40 Code SakSTAR(E80A, D82A, K130T, K135R) 20 250 8.4 17 3.111 13 18 4.3 11 5.5 1.5 <0.1 <0.1 <0.1 <0.1 1.0 80 64 68  SY6SakSTAR(K74R, E80A, D82A, K130T, K135R) 4 220 5.3 31 2.8 18 11 89 8.4 215.9 1.8 <0.1 <0.1 <0.1 <0.1 0.6 74 34 69 72  SY7 SakSTAR(K74Q, E80A,D82A, K130T, K135R) 27 110 5.1 6.5 1.2 7.9 7.3 28 6.4 19 4.6 1.7 <0.1<0.1 <0.1 <0.1 0.8 46 17 60 48 SY15 SakSTAR(K35A, K74R, E80A, D82A,K130T, 70 160 5.9 5.7 6.4 2.7 18 19 16 8.2 9.0 1.8 <0.1 <0.1 <0.1 <0.10.8 66 34 66 68 SY17 K135R) SakSTAR(E65D, K74R, E80A, D82A, K130T, 22140 5.4 50 2.9 49 21 31 11 37 3.4 1.8 <0.1 <0.1 <0.1 <0.1 0.6 43 11 6857 SY19 K135R) SakSTAR(E65S, K74R, E80A, D82A, K130T, 3 110 3.2 12 4.644 15 19 5.0 15 6.6 1.4 <0.1 <0.1 <0.1 <0.1 0.4 35 12 60 — SY20 K135R)SakSTAR(E65T, K74R, E80A, D82A, K130T, 30 94 7.2 9.3 9.9 5.6 32 22 158.2 11 2.0 <0.1 <0.1 <0.1 <0.1 1.0 58 24 69 — SY21 K135R) SakSTAR(S34G,S36R, K74R, K130T, K135R) 250 5.6 54 2.9 38 25 36 6.5 18 5.5 1.9 <0.1<0.1 <0.1 <0.1 0.9 75 33 68 — SY10 SakSTAR(E65A, K74R, E80A, D82A,K130T, 40 140 8.2 10 13 5.7 55 24 22 11 15 2.2 <0.1 <0.1 <0.1 <0.1 1.051 17 66 — SY18 K135R) SakSTAR(E65N, K74R, E80A, D82A, K130T, 88 120 8.512 11 7.0 36 43 13 12 7.9 2.4 <0.1 <0.1 <0.1 <0.1 0.9 60 29 67 — SY23K135R) SakSTAR(E65Q, K74R, E80A, D82A, K130T, 55 140 9.0 16 16 9.9 59 184.5 4.3 10 2.5 <0.1 <0.1 <0.1 <0.1 0.8 54 22 63 60 SY22 K135R)SakSTAR(K57A, E58A, E61A, E80A, D82A, 24 110 2.4 17 2.9 13 12 16 7.8 164.5 1.6 <0.1 <0.1 <0.1 <0.1 0.7 75 64 62 74 SY13 K130T, K135R)SakSTAR(E65A, A725, K74R, E80A, D82A, 92 62 8.2 23 3.8 8.4 19 30 11 133.6 3.1 <0.1 <0.1 <0.1 <0.1 1.2 51 13 66 52 SY53 K130T, K135R)SakSTAR(E65D, K74Q, E80A, D82A, K130T, 84 110 7.0 3.9 2.6 4.1 7.2 29 2814 6.8 2.2 <0.1 <0.1 <0.1 <0.1 0.9 43 13 64 42 SY30 K135R) SakSTAR(E65Q,K74Q, E80A, D82A, K130T, 54 120 5.1 16 3.2 14 3.7 16 20 2.4 5.7 1.7 <0.1<0.1 <0.1 <0.1 1.0 43 21 64 42 SY47 K135R) SakSTAR(K35A, E65D, K74Q,E80A, D82A, 56 140 5.1 6.8 2.6 9.5 1.7 28 17 14 8.4 2.1 <0.1 <0.1 <0.1<0.1 1.0 35 8 58 40 SY46 K130T, K135R) SakSTAR(K74R, E80A, D82A, S103A,32 160 4.9 22 5.8 14 4.3 26 6.9 7.4 5.0 1.7 <0.1 <0.1 <0.1 <0.1 0.9 6732 69 70 SY24 K130T, K135R) SakSTAR(K35A, E65D, K74R, E80A, D82A, 9.0 895.8 30 2.6 26 10 13 16 13 3.2 1.8 <0.1 <0.1 <0.1 <0.1 0.5 55 10 63 47SY12 E108A, K109A, K130T, K135R) SakSTAR(K35A, E65D, K74R, E80A, D82A,20 91 6.4 20 5.0 15 3.9 22 17 7.4 2.4 1.9 <0.1 <0.1 <0.1 <0.1 0.9 44 870 53 SY32 E108A, K130T, K135R) SakSTAR(E65D, K74R, E80A, D82A, 4 90 8.16.7 6.9 4.3 29 19 31 11 14 2.1 <0.1 <0.1 <0.1 <0.1 1.0 52 11 69 — SY33E108A, K130T, K135R) SakSTAR(K35A, E65D, K74R, E80A, D82A, 42 84 5.5 185.3 14 1.6 18 12 7.7 10 1.7 <0.1 <0.1 <0.1 <0.1 1.0 43 6 61 50 SY36K109A, K130T, K135R) SakSTAR(E65D, K74R, E80A, D82A, K109A, 60 130 9.76.6 6.8 4.2 28 11 32 12 17 2.3 <0.1 <0.1 <0.1 <0.1 0.9 56 10 64 53 SY37K130T, K135R) SakSTAR(K35A, E65D, K74R, E80A, D82A, 28 81 4.5 12 3.3 111.7 22 13 7.6 4.9 1.6 <0.1 <0.1 <0.1 <0.1 0.8 40 14 52 40 SY34 K130T,K135R, K136A) SakSTAR(E65D, K74R, E89A, D82A, K130T, 60 100 6.8 5.8 4.44.5 15 33 32 14 7.9 2.0 <0.1 <0.1 <0.1 <0.1 0.8 46 28 67 45 SY35 K135R,K136A) SakSTAR(E65Q, K74Q, D82A, S84A, K130T, 170 NT 45 21 60 45 SY50NK135R) SakSTAR(K35A, E65D, K74R, E80A, D82A, 68 86 4.4 20 5.5 15 1.5 1512 6.4 6.7 1.9 <0.1 <0.1 <0.1 <0.1 1.0 36 7 60 55 SY40 K86A, K130T,K135T) SakSTAR(K35A, K74Q, E80A, D82A, K130T, 72 120 6.1 3.4 2.5 3.0 5.938 14 9.8 6.8 1.9 <0.1 <0.1 <0.1 <0.1 0.8 49 16 64 48 SY28 K135R)SakSTAR(K35A, E65D, K74R, E80A, D82A, 54 190 8.1 7.5 6.9 5.5 25 37 34 147.7 2.3 <0.1 <0.1 <0.1 <0.1 1.0 56 28 68 55 SY29 K130T, K135R)SakSTAR(K35A, E65D, K74R, E80A, D82A, 13 55 6.7 23 5.3 17 2.3 47 19 195.1 2.0 <0.1 <0.1 <0.1 <0.1 1.1 53 20 88 62 SY61 V132R, K135R)SakSTAR(K35A, E65D, K74R, E80A, D82A, 13 61 7.0 13 5.1 31 12 27 12 116.7 2.5 <0.1 <0.1 <0.1 <0.1 1.9 56 18 79 60 SY62 T129A, K135R)SakSTAR(K35A, E65D, K74R, E80A, D82A, 23 21 6.9 27 5.8 32 20 29 6.6 9.75.4 2.1 <0.1 <0.1 <0.1 <0.1 0.9 56 17 91 60 SY64 T129A, K135A)Association constants ≧ 10-fold lower and antibody absorption ≦60percent of wild-type SakSTAR are represented in bold type; ≧ 100,000HU/mg represented in bold type. NT: not tested.

TABLE 8 SakSTAR variants with intact specific activity (≧100 kHU/mg) and≦50 percent absorption of human antibodies elicited by treatment withwild-type SakSTAR Spec. Act SakSTAR patient plasma Variant (kU/mg) Pool10 Subpool B Subpool C Pool 40 Code SakSTAR(K74Q, K130T, K135R) 190 5025 67 62 SY41 SakSTAR(E65A, K74Q, K130T, K135R) 170 45 16 77 55 SY48SakSTAR(E65Q, T71S, K74Q, K130T, K135R) 210 49 21 64 59 SY65SakSTAR(E65Q, K74Q, E118A, K130A, K135R) 180 50 28 72 58 SY73SakSTAR(E65Q, K74Q, N95A, E118A, K130A, K135R) 190 48 27 74 58 SY74SakSTAR(K35A, K65Q, K74Q, K130A, K35R) 110 49 26 63 45 SY75SakSTAR(E65Q, K74Q, K109A, K130T, K135R) 210 50 22 68 51 SY81SakSTAR(K74Q, E80A, D82A, K130T, K135R) 110 46 17 60 48 SY15SakSTAR(E65D, K74R, E80A, D82A, K30T, K135R) 140 43 11 68 57 SY19SakSTAR(E65S, K74R, E80A, D82A, K130T, K135R) 110 35 12 60 — SY20SakSTAR(E65D, K74R, E80A, D82A, K130T, K135R, K136A) 100 46 28 67 45SY35 SakSTAR(K35A, K74Q, E80A, D82A, K130T, K135R) 120 49 16 64 48 SY28SakSTAR(E65D, K74Q, E80A, D82A, K130T, K135R) 110 43 13 64 42 SY30SakSTAR(E65Q, K74Q, E80A, D82A, K130T, K135R) 120 43 21 64 42 SY47SakSTAR(E65Q, K74Q, D82A, S84A, K130T, K135R) 170 45 21 60 45 SY50NSakSTAR(K35A, E65D, K74Q, E80A, D82A, K130T, K135R) 140 35 8 58 40 SY46SakSTAR(T21A, K35A, E65Q, K74Q, K130A, K135R) 110 50 26 72 50 SY78SakSTAR(E65Q, K74Q, K109A, K121A, K130A, K135R) 140 50 31 73 52 SY88SakSTAR(E65Q, K74Q, D82A, S84A, K109A, K130A, K135R) 180 43 20 62 44SY89 SakSTAR(E65Q, K74Q, N95A, E118A, K130A, 120 45 30 74 60 SY93 K135R,+137A) SakSTAR(E65Q, K74Q, N95A, E118A, K130A, 1,400   37 16 70 54 SY94K135R, K136A, +137K) SakSTAR(E65Q, K74Q, D82A, S84A, E108A, 110 46 26 6341 SY95 K109A, K130A, K135R) Antibody absorption ≦60 percent ofwild-type SakSTAR are represented in bold type ≧100,000 HU/mgrepresented in bold type.

TABLE 9 Fibrinolytic properties of selected SakSTAR variants in humanplasma in vitro Residual fibrinogen Fibrinogenolytic Fibrinolyticpotency at C50 potency Compound (C50 in μg/mL) (% of baseline) (C50 inμg/mL) SakSTAR 0.18 ± 0.01 93 ± 3.5 24 ± 3.6 SakSTAR(K74Q, E80A, D82A,K130T, K135R) 0.15 ± 0.01 97 ± 3.0 14 ± 3.2 SakSTAR(E65D, K74R, E80A,D82A, K130T, K135R) 0.24 ± 0.04 94 ± 10  29 ± 3.1 SakSTAR(K35A, E65D,K74Q, E80A, D82A, K130T, K135R) 0.11 ± 0.01 92 ± 3.1 20 ± 2.0SakSTAR(E65Q, K74Q, N95A, E118A, K130A, K135R, K136A, +137K) 0.13 91 Thedata represent mean ± SD of 3 experiments. C50: amount of wild type orvariant SakSTAR required for 50% clot lysis or 50% fibrinogen breakdownin 2 hrs.

TABLE 10 Pharamacokinetic parameters of the disposition ofstaphylokinase-related antigen from plasma following bolus injection ofSakSTAR variants (100 μg/kg) in hamsters. C₀ A B t1/2(α) t1/2(β) V_(C)AUC Cl_(p) Variant (μg/mL) (μg/mL) (μg/mL) (min) (min) (mL) (μg × min ×mL⁻¹) (mL × min⁻¹) SakSTAR 0.8 ± 0.1 0.6 ± 0.1 0.2 ± 0.0 2.8 7.0 13 ±1.0 4.6 ± 0.4 2.2 ± 0.2 SakSTAR(K74Q, E80A, D82A, K130T, 0.5 ± 0.1 0.4 ±0.1 0.1 ± 0.0 2.0 10 20 ± 2.2 2.5 ± 0.3 4.1 ± 0.5 K135R) SakSTAR(E65D,K74R, E80A, D82A, 0.6 ± 0.0 0.5 ± 0.0 0.1 ± 0.0 2.0 10 16 ± 1.1 2.8 ±0.2 3.7 ± 0.3 K130T, K135R) SakSTAR(K35A, E65DK74Q, E80A, 1.1 ± 0.1 1.0± 0.1 0.1 ± 0.0 2.0 24 9.6 ± 0.7  6.4 ± 0.5 1.6 ± 0.1 D82A, K130T,K135R) Data are mean ± SEM of 4 experiments.

TABLE 11 Baseline characteristics and treatment outcome of the patientswith peripheral arterial occlusion treated with SakSTAR, SakSTAR(K74A,E80A, D82A, K130T, K135R) or SakSTAR(E65D, K74A, E80A, D82A, K130T,K135R) Total dose Total Age of Length of Recanal- of throm- durationCompound Gen- Age Clinical Locus of occlusion occlusion ization bybolytic a- of infu- Additional Patient Id. der (yrs) ischemia occlusion(days) (cm) thrombolysis gent (mg) sion (hrs) therapy SakSTAR PUT M 66Subacute Femoro-femoral 6 5 Complete 2 23 Stenting left IF graft arteryVERM M 73 Acute Right PA 2 6 Partial 13 23 Right upper leg amputationGEIV V 63 Restpain Left SFA 10 5 Complete 8 7 PTA POL M 46 SubacuteRight SFA 30 50 Partial 22 29 Lumbal sympath- ectomie BUE F 53Claudication Right AF graft 1 15 Complete 10 13 Desobstruction VIJ F 75Subacute Left FT graft 2 34 Complete 7 10 PTA REN M 48 Restpain Right IFgraft 4 20 Complete 6.5 5 Left AF graft COR V 78 Acute Left AFS 14 9Complete 4 3 PTA MAN M 67 Restpain Left tibial artery 1 6 Partial 6 5 —STRA M 66 Claudication Right FP graft 14 16 Complete 19 26 — VANH M 38Acute Left radial artery 4 1 Complete 6 5 — VANW F 57 Acute Right FPgraft 1 25 Complete 20 24 New right FP graft BRA M 57 Acute Left FTgraft 1 30 Complete 25 43 — DON M 60 Claudication Left FT graft 1 20Complete 13 19 PTA + stenting CAM M 77 Restpain Right SFA graft 8 30Complete 27 44 FF graft Mean ± SEM 62 ± 3.1 6.6 ± 2.1 18 ± 3.5 13 ± 2.119 ± 3.5 SakSTAR(K74Q, E80A, D82A, K130T, K135R) IMB M 66 ClaudicationLeft SFA 30 5 Complete 24 24 PTA AZY M 44 Subacute Right C.I.A, 7 8Complete 18 23 Stenting VIN M 51 Acute Right E.I.A, 5 70 Complete 24 30— STRO M 53 Claudication Left FP junction 14 5 Partial 3.5 2 Aspirationthrom- bectomy, PTA VERG M 62 Restpain Left SFA 20 6 Complete 19 25 FPbypass GIE M 76 Acute Right FP bypass 2 15 Complete 8.5 17 — Mean ± SEM59 ± 4.7 13 ± 4.3 18 ± 10 16 ± 3.4 20 ± 4.0 SakSTAR(E65D, K74R, E80A,D82A, K130T, K135R) URB M 57 Subacute Right E.I.A, 4 8 Complete 8 6Pseudo aneutysm, right AF graft revision COM M 59 Restpain Right AFgraft 7 65 Complete 16 22 — HAC M 70 Restpain Left anterior tibial 7 15Complete 12 14 artery DEW F 76 Restpain SFA 21 6 Complete 6 4 — VAI F 65Subacute Left PA 25 10 Partial 8 6 Aspiration throm- bectomy FIL M 76Claudication Right SFA 28 8 Complete 24 31 PTA Mean ± SEM 67 ± 3.4 15 ±4.3 19 ± 9.4 12 ± 2.8 14 ± 4.4 AF: aortofemoral; CABG: coronary arterybypass graft; CAD, coronary artery disease; CIA: common iliac artery;COPD: chronic obstructive pulmonary disease; DM: diabetes mellitus; EIA;external iliac artery; FF: femorofibular; FP: femoropopliteal; FT:femorotibial; IA: iliac artery; IF: iliofemoral; occl: occlusion; PA:popliteal artery; PTA: percutaneous transluminal angioplasty; SFA,superficial femoral artery; Ta: tibial artery; TF: tibiofibular; SC:subclavian.

TABLE 12 Absorption with SakSTAR variants of antibodies elicited withSakSTAR variants in patients with peripheral arterial occlusionInsolubilized compound SakSTAR(K74Q, SakSTAR(E65D, K74R, E80A, D82A,E80A, D82A, Treatment Absorbant SakSTAR K130T, K135R) K130T, K135R)SakSTAR SakSTAR 95 (Pool 40) SakSTAR(K74Q, E80A, D82A, K130T, K135R) 48SalSTAR(E65D, K74R, E80A, D82A, K130T, K135R) 57 SakSTAR(K74Q, E80A,D82A, SakSTAR 94 95 95 K130T, K135R) SakSTAR(K74Q, E80A, D82A, K130T,K135R) 91 93 89 (Imb., Vin., Ver., Gie.) SalSTAR(E65D, K74R, E80A, D82A,K130T, K135R) 92 94 94 SakSTAR(E65D, K74R, SakSTAR 90 88 85 E80A, D82A,K130T, SakSTAR(K74Q, E80A, D82A, K130T, K135R) 94 95 94 K135R) (Urb.)SalSTAR(E65D, K74R, E80A, D82A, K130T, K135R) 94 95 94 Data representmedian values of the percent absorption with 250 nM absorbant, measuredby residual binding to insolubilized compound. *p= . . . versus SakSTAR;**p= . . . versus SakSTAR(K74Q, E80A, D82A, K130T, K135R)); and p= . . .versus SakSTAR(E65D, K74R, E80A, D82A, K130T, K135R) by pairednonparametric test.

TABLE 13 Cysteine substitution variants of SakSTAR Clot lysis in vitroSpec. Act. (C₅₀ in t1/2(α) Clp Variant (kU/mg) Dimerization level (%)PEG derivatization μg/ml) (min) (ml/min) SakSTAR 130 0 none 0.33 2.02.2  SakSTAR (K102C) 143 0 none 0.29 nd nd SakSTAR (K102C-PEG) 108 0 10.60 3.0 0.32 SakSTAR (K109C) monomeric 100 0 none 0.52 nd nd SakSTAR(K109C) dimeric 1,650   >60 none 0.17 3.6 0.52 2,235   >95 none 0.12 ndnd

10 1 20 DNA Staphylokinase aureus 1 caggaaacag aattcaggag 20 2 28 DNAStaphylokinase aureus 2 caaaacagcc aagcttcatt cattcagc 28 3 28 DNAArtificial Sequence Staphylococcus aureus staphylokinase 3′ PCRselection-primer LY34 3 caaaacagcc gagcttcatt cattcagc 28 4 41 DNAArtificial Sequence Staphylococcus aureus staphylokinase mutagenic PCRprimer LY58 4 ttcagcatgc tgcagttatt tcttttctgc aacaaccttg g 41 5 27 DNAStaphylococcus aureus 5 caaacagcca agcttcattc attcagc 27 6 27 DNAArtificial Sequence Staphylococcus aureus staphylokinase SOE-PCRbackward primer for construction of mutant K102C 6 tatgataaga attgcaaaaaagaagaa 27 7 27 DNA Artificial Sequence Staphylococcus aureusstaphylokinase SOE-PCR forward primer for construction of mutant K102C 7ttcttctttt ttgcaattct tatcata 27 8 27 DNA Artificial SequenceStaphylococcus aureus staphylokinase SOE-PCR backward primer forconstruction of mutant K109C 8 aaaaagaaga aacgtgctct ttcccta 27 9 27 DNAArtificial Sequence Staphylococcus aureus staphylokinase SOE-PCR forwardprimer for construction of mutant K109C 9 tagggaaaga gcacgtttct tcttttt27 10 136 PRT Staphylococcus aureus 10 Ser Ser Ser Phe Asp Lys Gly LysTyr Lys Lys Gly Asp Asp Ala Ser 1 5 10 15 Tyr Phe Glu Pro Thr Gly ProTyr Leu Met Val Asn Val Thr Gly Val 20 25 30 Asp Ser Lys Gly Asn Glu LeuLeu Ser Pro His Tyr Val Glu Phe Pro 35 40 45 Ile Lys Pro Gly Thr Thr LeuThr Lys Glu Lys Ile Glu Tyr Tyr Val 50 55 60 Glu Trp Ala Leu Asp Ala ThrAla Tyr Lys Glu Phe Arg Val Val Glu 65 70 75 80 Leu Asp Pro Ser Ala LysIle Glu Val Thr Tyr Tyr Asp Lys Asn Lys 85 90 95 Lys Lys Glu Glu Thr LysSer Phe Pro Ile Thr Glu Lys Gly Phe Val 100 105 110 Val Pro Asp Leu SerGlu His Ile Lys Asn Pro Gly Phe Asn Leu Ile 115 120 125 Thr Lys Val ValIle Glu Lys Lys 130 135

What is claimed is:
 1. A staphylokinase derivative comprising an aminoacid sequence which differs from SEQ ID NO:10 due to substitution of oneor more amino acids therein with cysteine thus reducing the clearancefrom plasma in patients, and further wherein the staphylokinasederivative has a fibrinolytic or fibrinogenolytic property.
 2. Thestaphylokinase derivative of claim 1, in which the substitution furtherresults in dimerization, increased specific activity, reduced clearanceand/or increased thrombolytic potency of the derivative.
 3. Thestaphylokinase derivatives as claimed in claim 1, in which amino acidK109 has been replaced by a cysteine.
 4. A dimeric staphylokinasederivative comprising a peptide having essentially the amino acidsequence depicted in FIG. 1 (SEQ ID NO: 10) in which amino acid K109 hasbeen replaced by a cysteine.
 5. A pharmaceutical composition comprisingat least one of the staphylokinase derivatives as claimed in claim 2together with a suitable excipient.